Methods to treat cancer with cupredoxins

ABSTRACT

The present invention discloses methods and materials for killing and/or inhibiting the growth of a cancer cell via preferential entry of a cytotoxic compound. Preferential entry of the cytotoxic compound is accomplished by the use of protein transduction domains derived from cupredoxins, including the p18 and p28 truncations of azurin.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §§ 119 and 120 to andis a continuation in part of U.S. patent application Ser. No.12/314,703, filed on Dec. 15, 2008 now abandoned, which claims priorityto U.S. Patent Application Ser. No. 61/013,709, filed on Dec. 14, 2007;and is a continuation in part of Ser. No. 12/028,683, filed Feb. 8, 2008now U.S. Pat. No. 8,232,244, which claims priority to U.S. PatentApplication Ser. No. 60/900,098, filed Feb. 8, 2007; and is acontinuation in part of application Ser. No. 11/488,693, filed Jul. 19,2006 now U.S. Pat. No. 7,556,810, which claims priority to U.S. PatentApplication Ser. No. 60/700,297, filed Jul. 19, 2005; and is acontinuation in part of U.S. patent application Ser. No. 11/244,105,filed Oct. 6, 2005 now U.S. Pat. No. 7,691,383, which claims priority toU.S. Provisional Patent Application Ser. No. 60/616,782, filed Oct. 7,2004, and U.S. Provisional Patent Application Ser. No. 60/680,500, filedMay 13, 2005.

This application contains one (1) disc containing a sequence listing.The materials recorded in the compact disc are incorporated herein byreference in their entirety. The compact disc contains a single filenamed “14PR1418.txt” (49 KB, created on Feb. 5, 2009). The compact discwas created on Feb. 5, 2009.

FIELD OF THE INVENTION

The present invention relates to compositions comprising cupredoxins,and variants, derivatives and structural equivalents of cupredoxins thatinhibit the development of premalignant lesions in mammalian cells,tissues and animals. The invention also relates to the use ofcupredoxins, and variants, derivatives and structurally equivalents ofcupredoxins, as chemopreventive agents in mammals to inhibit thedevelopment of premalignant lesions, and ultimately cancer.

BACKGROUND

Cancer chemoprevention is the use of natural, synthetic or biologicchemical agents to reverse, suppress, or prevent carcinogenicprogression to invasive cancer. Recent clinical trials in preventingcancer in high-risk populations suggest that chemopreventive therapy isa realistic treatment for high-risk patients. Chemopreventive therapy isbased on the concepts of multifocal field carcinogenesis and multistepcarcinogenesis. In field carcinogenesis, generalized carcinogen exposurethroughout the tissue field results in diffuse epithelial injury intissue and clonal proliferation of the mutated cells. These geneticmutations throughout the field increase the likelihood that one or morepremalignant or malignant lesions may develop in the field. Multistepcarcinogenesis in the stepwise accumulation of these genetic andphenotypic alterations. Arresting one or more steps in the multistepcarcinogenesis may impede or prevent the development of cancer. Seegenerally Tsao et al., CA Cancer J Clin 54:150-180 (2004).

The mouse mammary gland organ culture (MMOC) assay may be used toevaluate the inhibitory effects of potential chemopreventive agents onboth hormone-induced structural differentiation of mammary glands and onthe development of DMBA-induced prencoplastic hyperplastic alveolarmodule-like lesions in the gland. Mammary glands from young, virginanimals, when incubated for 6 days in the presence of insulin(I)+prolactin (P)+aldosterone (A), can differentiate into fully-grownglands. These glands morphologically resemble the glands obtained frompregnant mice. Aldosterone can be replaced by estrogen (E)+progesterone(Pg) Inclusion of hydrocortisone (H) to the medium stimulates thefunctional differentiation of the mammary glands. Mehta and Banerjee,Acta Endocrinol. 80:501 (1975); Mehta and Moon, Breast Cancer: Treatmentand Prognosis 300, 300 (Basil A Stoll ed., Blackwell Press 1986). Thus,the hormone-induced structural and functional differentiation, observedin this culture system, mimics the responses to hormones observed duringvarious physiological stages of the animal.

Mice exhibit a distinct preneoplastic stage prior to cancer formation inMMOC. Such preneoplastic lesions in C3H mice are induced by murinemammary tumor virus or in BALB/c mice by DMBA. Exposure of the glands to2 μg/ml DMBA between days 3 and 4 of growth phases followed byregression of the glands for 2-3 weeks in the medium containing onlyinsulin, results in the formation of mammary alveolar lesions (MAL).Hawthorne et al., Pharmaceutical Biology 40:70-74 (2002); Mehta et al.,Methods in Cell Science 19:19-24 (1997). Furthermore, transplantation ofepithelial cells, prepared from glands containing the DMBA-inducedmammary lesions, into syngeneic host resulted in the development ofmammary adenocarcinoma. Telang et al., PNAS 76:5886-5890 (1979).Pathologically, these tumors were similar to those observed in vivo whenmice of the same strain are administered DMBA. Id.

DMBA-induced mammary lesion formation in MMOC can be inhibited by avariety of classes of chemopreventive agents such as retinoids. Theseagents include chemopreventive agents derived from the natural productssuch as brassinin and resveretrol, thiols, antioxidants, inhibitors ofornithine decarboxylase such as OFMO and deguelin, inhibitors ofprostaglandin synthesis, Ca regulators, etc. Jang et al., Science275:218-220 (1997); Mehta, Eur. J. Cancer 36:1275-1282 (2000); Metha etal., J. Natl. Cancer Inst. 89:212-219 (1997). These studies clearlydemonstrate that this organ culture system offers a unique model todetermine the effectiveness of compounds against mammary carcinogenesis.The results can be expected to closely correlate to the inhibitionobtained by in vivo administration of such compounds.

The MMOC may also be induced to form mammary ductal lesions (MDL). TheMDL can be induced if estrogen and progesterone instead of aldosteroneand hydrocortisone are included in the medium. The alveolar structuresin the presence of ovarian steroids are very small but the intraductallesions are observed in histopathological sections. Mehta et al., J.Natl. Cancer Inst. 93:1103-1106 (2001). The antiestrogens, whichselectively work on ovarian hormone dependent ER+breast cancers such astamoxifen, inhibited MDL formation and not MAL. Thus, this modifiedculture model in addition to conventional MAL induction protocol now canbe used to evaluate effects of chemopreventive agents on both MAL andMOL.

The entry of a protein into a mammalian cell is often dictated by asmall segment of the protein, which is commonly referred to as a“protein transduction domain” or PTD. This segment can be used as asignal attached to a foreign protein to facilitate transport of such aprotein into a mammalian cell. For example, amphipathic peptides areused to facilitate uptake of DNA-cleaving metalloporphyrins as potentialantitumor drugs in human fibroblasts HS68 or murine lymphocytic leukemiaL₁₂₁₀ cells (Chaloin, L. et al Bioconjugate Chem. 12:691-700, (2001)).

Peptides called cell-penetrating peptides (CPPs) or cell-deliveryvectors (CDVs), such as penetratin, transportan, Tat (amino acids 47-57or 48-60), and the model amphipathic peptide MAP, are short, amphipathicand cationic peptides and peptide derivatives, usually containingmultiple lysine and arginine residues. Fischer, P. M., Med Res Rev, 27:755-795 (2007). They form a class of small molecules receivingsignificant attention as potential transport agents or delivery vehiclesfor a variety of cargoes, including cytotoxic drugs, anti-senseoligo-nucleotides, proteins, and peptides, in gene therapy, and as decoypeptides. Hallbrink, M. et al. Biochim. Biophys. Acta 1515: 101-109(2001); Lindgren, M., et al. Trends Pharmacol. Sci. 21: 99-103 (2000);Gusarova, et al, J Clin Invest, 117: 99-111 (2007); Melnick, A., BiochemSoc Trans, 35: 802-806 (2007); Astriab-Fisher et al., Pharm Res, 19:744-754 (2002); El-Andaloussi et al., J Gene Med, 8: 1262-1273 (2006);Cashman et al., Mol Ther, 6: 813-823 (2002).

SUMMARY OF THE EMBODIMENTS

The present invention relates to compositions and methods comprisingpeptides that may be cupredoxins or variants, derivatives and structuralequivalents of cupredoxins that preferentially enter cells and alsoinhibit the development of premalignant lesions in mammalian cells,tissues and animals.

The present invention further relates to methods comprising killing acancer cell by contacting the cancer cell with a cytotoxic cupredoxin,wherein the cytotoxic cupredoxin preferentially enters the cancer cellvia one or more endocytotic pathways, and wherein the cytotoxiccupredoxin is a truncation of azurin, and wherein the truncation ofazurin comprises one or more of the amino acids from the C-terminus ofSEQ ID NO: 2. In some embodiments, the truncation of azurin is fromPseudomonas aeruginosa. In other embodiments, the truncation of azurincomprises SEQ ID NO: 2. In yet other embodiments, the truncation ofazurin consists of SEQ ID NO: 2.

In another embodiment, the cytotoxic cupredoxin preferentially entersthe cancer cell via caveolae-mediated endocytosis. In anotherembodiment, the entry of the cytotoxic cupredoxin into the cancer cellis mediated by the Golgi apparatus.

In a further embodiment, the cytotoxic cupredoxin comprises amino acidscapable of contacting the cell membrane of the cancer cell irrespectiveof the cancer cell's status. In some embodiments, the cytotoxiccupredoxin contacts amino acids, cell surface peptides, and/or receptorson the cell membrane. In some embodiments, the cytotoxic cupredoxin maycomprise each of the amino acids located at positions 69, 70, 75, 76,and 85 of SEQ ID NO: 1. In another embodiment, the cytotoxic cupredoxincomprises one or more of the amino acids located at positions 69, 70,75, 76, and 85 of SEQ ID NO: 1. In any of these embodiments, these aminoacids may be located at positions within the cytotoxic cupredoxinsimilar or homologous to those of SEQ ID NO: 1.

In another embodiment, the cytotoxic cupredoxin comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO:36, and SEQ ID NO: 37. In another embodiment, the cytotoxic cupredoxinconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37.

The present invention further relates to isolated peptides capable ofcontacting the cell membrane of a cancer cell, entering the cancer cellvia caveolae-mediated endocytosis, and killing the cancer cell, whereinthe isolated peptides comprise the C-terminal amino acids of SEQ ID NO:2. In some embodiments, the isolated peptide contacts amino acids, cellsurface peptides, and/or receptors on the cell membrane. In someembodiments, the entry of the isolated peptide into the cancer cell ismediated by the Golgi apparatus. In other embodiments, the isolatedpeptide is from Psuedomonas aeruginosa. In further embodiments, theisolated peptide comprises SEQ ID NO: 2. In other embodiments, theisolated peptide consists of SEQ ID NO: 2. In yet other embodiments, theisolated peptide consists of the C-terminal amino acids of SEQ ID NO: 2.For example, the isolated peptide may comprise an amino acid sequenceselected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, andSEQ ID NO: 37 SEQ ID NO: 36. In another embodiment, the isolated peptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37 SEQ ID NO: 36.

The present invention also relates to isolated peptides capable ofcontacting the cell membrane of a cancer cell, entering the cancer cellvia caveolae-mediated endocytosis, and killing the cancer cell, whereinthe isolated peptides comprise one or more of the amino acids found atpositions 69, 70, 75, 76, and 85 of SEQ ID NO: 1. In furtherembodiments, an isolated peptide comprises each of the amino acidslocated at positions 69, 70, 75, 76, and 85 of SEQ ID NO: 1. In theseembodiments, these amino acids may be located at positions within theisolated peptide similar or homologous to those of SEQ ID NO: 1. In someembodiments, the isolated peptide contacts amino acids, cell surfacepeptides, and/or receptors on the cell membrane.

The invention further relates to a pharmaceutical composition comprisingone or more of the isolated peptides described above. In someembodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier. In other embodiments, in thepharmaceutically acceptable carrier is suitable for intravenousadministration.

The invention further relates to a method comprising treating amammalian patient by administering to the patient a therapeuticallyeffective amount of one or more of the pharmaceutical compositions ofthe invention. In some embodiments, the patient is human. In otherembodiments, the patient is at a higher risk to develop cancer than thegeneral population. In further embodiments, the cancer is selected frommelanoma, breast, pancreas, glioblastoma, astrocytoma, lung, colorectal,neck and head, bladder, prostate, skin, and cervical cancer. In otherembodiments, the patient has at least one high risk feature. In anotherembodiment, the patient has premalignant lesions. In another embodiment,the patient has been cured of cancer or premalignant lesions. In someembodiments, the pharmaceutical composition is administered by a modeselected from the group consisting of intravenous injection,intramuscular injection, subcutaneous injection, inhalation, topicaladministration, transdermal patch, suppository, vitreous injection andoral. In a specific embodiment, the mode of administration is byintravenous injection.

The invention further relates to a kit comprising one or more of thepharmaceutical compositions of the invention in a vial. In someembodiments, the kit further comprises an apparatus to administer theactive composition to a patient.

The invention further relates to a pharmaceutical composition comprisingone or more of the isolated peptides of the invention and a cargocompound. In some embodiments, the isolated peptide is linked to thecargo compound. In one specific embodiment, the cargo compound isTamoxifen. In other embodiments, the cargo compound is selected from thegroup consisting of a protein, lipoprotein, polypeptide, peptide,polysaccharide, nucleic acid, dye, microparticle, nanoparticle, toxin,and drug. In another embodiment, the cargo compound is a detectablesubstance. For example, the cargo compound may be an X-ray contrastagent detectable by X-ray CT, a magnetic resonance imaging contrastagent detectable by MRI, or an ultrasound contrast agent and isdetectable by ultrasound. In some embodiments, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable carrier.

The invention also relates to a method comprising delivering a cargocompound into a cell by contacting the cell with a pharmaceuticalcomposition comprising one or more of the isolated peptides describedabove and a cargo compound, as described above.

These and other aspects, advantages, and features of the invention willbecome apparent from the following figures and detailed description ofthe specific embodiments.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1. Amino acid sequence of azurin from Pseudomonas aeruginosa(Ala Glu Cys Ser Val Asp Ile Gln Gly Asn Asp GlnMet Gln Phe Asn Thr Asn Ala Ile Thr Val Asp LysSer Cys Lys Gln Phe Thr Val Asn Leu Ser His ProGly Asn Leu Pro Lys Asn Val Met Gly His Asn TrpVal Leu Ser Thr Ala Ala Asp Met Gln Gly Val ValThr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp TyrLeu Lys Pro Asp Asp Ser Arg Val Ile Ala His ThrLys Leu Ile Gly Ser Gly Glu Lys Asp Ser Val ThrPhe Asp Val Ser Lys Leu Lys Glu Gly Glu Gln TyrMet Phe Phe Cys Thr Phe Pro Gly His Ser Ala LeuMet Lys Gly Thr Leu Thr Leu Lys).SEQ ID NO: 2. Amino acid sequence of p28,Pseudomonas aeruginosa azurin residues 50-77(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 3. Amino acid sequence of plastocyaninfrom Phormidium laminosum(Glu Thr Phe Thr Val Lys Met Gly Ala Asp Ser GlyLeu Leu Gln Phe Glu Pro Ala Asn Val Thr Val HisPro Gly Asp Thr Val Lys Trp Val Asn Asn Lys LeuPro Pro His Asn Ile Leu Phe Asp Asp Lys Gln ValPro Gly Ala Ser Lys Glu Leu Ala Asp Lys Leu SerHis Ser Gln Leu Met Phe Ser Pro Gly Glu Ser TyrGlu Ile Thr Phe Ser Ser Asp Phe Pro Ala Gly ThrTyr Thr Tyr Tyr Cys Ala Pro His Arg Gly Ala GlyMet Val Gly Lys Ile Thr Val Glu Gly).SEQ ID NO: 4. Amino acid sequence of rusticyaninfrom Thiobacillus ferrooxidans(Gly Thr Leu Asp Thr Thr Trp Lys Glu Ala Thr LeuPro Gln Val Lys Ala Met Leu Glu Lys Asp Thr GlyLys Val Ser Gly Asp Thr Val Thr Tyr Ser Gly LysThr Val His Val Val Ala Ala Ala Val Leu Pro GlyPhe Pro Phe Pro Ser Phe Glu Val His Asp Lys LysAsn Pro Thr Leu Glu Ile Pro Ala Gly Ala Thr ValAsp Val Thr Phe Ile Asn Thr Asn Lys Gly Phe GlyHis Ser Phe Asp Ile Thr Lys Lys Gly Pro Pro TyrAla Val Met Pro Val Ile Asp Pro Ile Val Ala GlyThr Gly Phe Ser Pro Val Pro Lys Asp Gly Lys PheGly Tyr Thr Asp Phe Thr Trp His Pro Thr Ala GlyThr Tyr Tyr Tyr Val Cys Gln Ile Pro Gly His AlaAla Thr Gly Met Phe Gly Lys Ile Val Val Lys).SEQ ID NO: 5. Amino acid sequence of pseudoazurinfrom Achromobacter cycloclastes(Ala Asp Phe Glu Val His Met Leu Asn Lys Gly LysAsp Gly Ala Met Val Phe Glu Pro Ala Ser Leu LysVal Ala Pro Gly Asp Thr Val Thr Phe Ile Pro ThrAsp Lys Gly His Asn Val Glu Thr Ile Lys Gly MetIle Pro Asp Gly Ala Gln Ala Phe Lys Ser Lys IleAsn Glu Asn Tyr Lys Val Thr Phe Thr Ala Pro GlyVal Tyr Gly Val Lys Cys Thr Pro His Tyr Gly MetGly Met Val Gly Val Val Gln Val Gly Asp Ala ProAla Asn Leu Glu Ala Val Lys Gly Ala Lys Asn ProLys Lys Ala Gln Glu Arg Leu Asp Ala Ala Leu Ala Ala Leu Gly Asn).SEQ ID NO: 6. Amino acid sequence of azurin from Alcaligenes faecalis(Ala Cys Asp Val Ser Ile Glu Gly Asn Asp Ser MetGln Phe Asn Thr Lys Ser Ile Val Val Asp Lys ThrCys Lys Glu Phe Thr Ile Asn Leu Lys His Thr GlyLys Leu Pro Lys Ala Ala Met Gly His Asn Val ValVal Ser Lys Lys Ser Asp Glu Ser Ala Val Ala ThrAsp Gly Met Lys Ala Gly Leu Asn Asn Asp Tyr ValLys Ala Gly Asp Glu Arg Val Ile Ala His Thr SerVal Ile Gly Gly Gly Glu Thr Asp Ser Val Thr PheAsp Val Ser Lys Leu Lys Glu Gly Glu Asp Tyr AlaPhe Phe Cys Ser Phe Pro Gly His Trp Ser Ile MetLys Gly Thr Ile Glu Leu Gly Ser).SEQ ID NO: 7. Amino acid sequence of azurin fromAchromobacter xylosoxidans ssp. denitrificans I(Ala Gln Cys Glu Ala Thr Ile Glu Ser Asn Asp AlaMet Gln Tyr Asn Leu Lys Glu Met Val Val Asp LysSer Cys Lys Gln Phe Thr Val His Leu Lys His ValGly Lys Met Ala Lys Val Ala Met Gly His Asn TrpVal Leu Thr Lys Glu Ala Asp Lys Gln Gly Val AlaThr Asp Gly Met Asn Ala Gly Leu Ala Gln Asp TyrVal Lys Ala Gly Asp Thr Arg Val Ile Ala His ThrLys Val Ile Gly Gly Gly Glu Ser Asp Ser Val ThrPhe Asp Val Ser Lys Leu Thr Pro Gly Glu Ala TyrAla Tyr Phe Cys Ser Phe Pro Gly His Trp Ala MetMet Lys Gly Thr Leu Lys Leu Ser Asn).SEQ ID NO: 8. Amino acid sequence of azurin fromBordetella bronchiseptica(Ala Glu Cys Ser Val Asp Ile Ala Gly Thr Asp GlnMet Gln Phe Asp Lys Lys Ala Ile Glu Val Ser LysSer Cys Lys Gln Phe Thr Val Asn Leu Lys His ThrGly Lys Leu Pro Arg Asn Val Met Gly His Asn TrpVal Leu Thr Lys Thr Ala Asp Met Gln Ala Val GluLys Asp Gly Ile Ala Ala Gly Leu Asp Asn Gln TyrLeu Lys Ala Gly Asp Thr Arg Val Leu Ala His ThrLys Val Leu Gly Gly Gly Glu Ser Asp Ser Val ThrPhe Asp Val Ala Lys Leu Ala Ala Gly Asp Asp TyrThr Phe Phe Cys Ser Phe Pro Gly His Gly Ala LeuMet Lys Gly Thr Leu Lys Leu Val Asp).SEQ ID NO: 9. Amino acid sequence of azurin from Methylomonas sp. J(Ala Ser Cys Glu Thr Thr Val Thr Ser Gly Asp ThrMet Thr Tyr Ser Thr Arg Ser Ile Ser Val Pro AlaSer Cys Ala Glu Phe Thr Val Asn Phe Glu His LysGly His Met Pro Lys Thr Gly Met Gly His Asn TrpVal Leu Ala Lys Ser Ala Asp Val Gly Asp Val AlaLys Glu Gly Ala His Ala Gly Ala Asp Asn Asn PheVal Thr Pro Gly Asp Lys Arg Val Ile Ala Phe ThrPro Ile Ile Gly Gly Gly Glu Lys Thr Ser Val LysPhe Lys Val Ser Ala Leu Ser Lys Asp Glu Ala TyrThr Tyr Phe Cys Ser Tyr Pro Gly His Phe Ser MetMet Arg Gly Thr Leu Lys Leu Glu Glu).SEQ ID NO: 10. Amino acid sequence of azurin fromNeisseria meningitidis Z2491(Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu AlaThr Pro Ala Ala Glu Ala Pro Ala Ser Glu Ala ProAla Ala Glu Ala Ala Pro Ala Asp Ala Ala Glu AlaPro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu SerAsn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile GlnVal Ser Lys Ala Cys Lys Glu Phe Thr Ile Thr LeuLys His Thr Gly Thr Gln Pro Lys Thr Ser Met GlyHis Asn Ile Val Ile Gly Lys Thr Glu Asp Met AspGly Ile Phe Lys Asp Gly Val Gly Ala Ala Asp ThrAsp Tyr Val Lys Pro Asp Asp Ala Arg Val Val AlaHis Thr Lys Leu Ile Gly Gly Gly Glu Glu Ser SerLeu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly GluTyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly AlaLeu Met Asn Gly Lys Val Thr Leu Val Asp).SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonas fluorescen(Ala Glu Cys Lys Thr Thr Ile Asp Ser Thr Asp GlnMet Ser Phe Asn Thr Lys Ala Ile Glu Ile Asp LysAla Cys Lys Thr Phe Thr Val Glu Leu Thr His SerGly Ser Leu Pro Lys Asn Val Met Gly His Asn LeuVal Ile Ser Lys Gln Ala Asp Met Gln Pro Ile AlaThr Asp Gly Leu Ser Ala Gly Ile Asp Lys Asn TyrLeu Lys Glu Gly Asp Thr Arg Val Ile Ala His ThrLys Val Ile Gly Ala Gly Glu Lys Asp Ser Leu ThrIle Asp Val Ser Lys Leu Asn Ala Ala Glu Lys TyrGly Phe Phe Cys Ser Phe Pro Gly His Ile Ser MetMet Lys Gly Thr Val Thr Leu Lys).SEQ ID NO: 12. Amino acid sequence of azurin fromPseudomonas chlororaphis(Ala Glu Cys Lys Val Asp Val Asp Ser Thr Asp GlnMet Ser Phe Asn Thr Lys Glu Ile Thr Ile Asp LysSer Cys Lys Thr Phe Thr Val Asn Leu Thr His SerGly Ser Leu Pro Lys Asn Val Met Gly His Asn TrpVal Leu Ser Lys Ser Ala Asp Met Ala Gly Ile AlaThr Asp Gly Met Ala Ala Gly Ile Asp Lys Asp TyrLeu Lys Pro Gly Asp Ser Arg Val Ile Ala His ThrLys Ile Ile Gly Ser Gly Glu Lys Asp Ser Val ThrPhe Asp Val Ser Lys Leu Thr Ala Gly Glu Ser TyrGlu Phe Phe Cys Ser Phe Pro Gly His Asn Ser MetMet Lys Gly Ala Val Val Leu Lys).SEQ ID NO: 13. Amino acid sequence of azurin fromXylella fastidiosa 9a5c (Lys Thr Cys Ala Val Thr Ile Ser Ala Asn Asp GlnMet Lys Phe Asp Gln Asn Thr Ile Lys Ile Ala AlaGlu Cys Thr His Val Asn Leu Thr Leu Thr His ThrGly Lys Lys Ser Ala Arg Val Met Gly His Asn TrpVal Leu Thr Lys Thr Thr Asp Met Gln Ala Val AlaLeu Ala Gly Leu His Ala Thr Leu Ala Asp Asn TyrVal Pro Lys Ala Asp Pro Arg Val Ile Ala His ThrAla Ile Ile Gly Gly Gly Glu Arg Thr Ser Ile ThrPhe Pro Thr Asn Thr Leu Ser Lys Asn Val Ser TyrThr Phe Phe Cys Ser Phe Pro Gly His Trp Ala LeuMet Lys Gly Thr Leu Asn Phe Gly Gly).SEQ ID NO: 14. Amino acid sequence of stellacyanin from Cucumis sativus(Met Gln Ser Thr Val His Ile Val Gly Asp Asn ThrGly Trp Ser Val Pro Ser Ser Pro Asn Phe Tyr SerGln Trp Ala Ala Gly Lys Thr Phe Arg Val Gly AspSer Leu Gln Phe Asn Phe Pro Ala Asn Ala His AsnVal His Glu Met Glu Thr Lys Gln Ser Phe Asp AlaCys Asn Phe Val Asn Ser Asp Asn Asp Val Glu ArgThr Ser Pro Val Ile Glu Arg Leu Asp Glu Leu GlyMet His Tyr Phe Val Cys Thr Val Gly Thr His CysSer Asn Gly Gln Lys Leu Ser Ile Asn Val Val AlaAla Asn Ala Thr Val Ser Met Pro Pro Pro Ser SerSer Pro Pro Ser Ser Val Met Pro Pro Pro Val MetPro Pro Pro Ser Pro Ser).SEQ ID NO: 15. Amino acid sequence of auracyanin Afrom Chloroflexus aurantiacus(Met Lys Ile Thr Leu Arg Met Met Val Leu Ala ValLeu Thr Ala Met Ala Met Val Leu Ala Ala Cys GlyGly Gly Gly Ser Ser Gly Gly Ser Thr Gly Gly GlySer Gly Ser Gly Pro Val Thr Ile Glu Ile Gly SerLys Gly Glu Glu Leu Ala Phe Asp Lys Thr Glu LeuThr Val Ser Ala Gly Gln Thr Val Thr Ile Arg PheLys Asn Asn Ser Ala Val Gln Gln His Asn Trp IleLeu Val Lys Gly Gly Glu Ala Glu Ala Ala Asn IleAla Asn Ala Gly Leu Ser Ala Gly Pro Ala Ala AsnTyr Leu Pro Ala Asp Lys Ser Asn Ile Ile Ala GluSer Pro Leu Ala Asn Gly Asn Glu Thr Val Glu ValThr Phe Thr Ala Pro Ala Ala Gly Thr Tyr Leu TyrIle Cys Thr Val Pro Gly His Tyr Pro Leu Met GlnGly Lys Leu Val Val Asn).SEQ ID NO: 16. Amino acid sequence of auracyanin Bfrom Chloroflexus aurantiacus(Ala Ala Asn Ala Pro Gly Gly Ser Asn Val Val AsnGlu Thr Pro Ala Gln Thr Val Glu Val Arg Ala AlaPro Asp Ala Leu Ala Phe Ala Gln Thr Ser Leu SerLeu Pro Ala Asn Thr Val Val Arg Leu Asp Phe ValAsn Gln Asn Asn Leu Gly Val Gln His Asn Trp ValLeu Val Asn Gly Gly Asp Asp Val Ala Ala Ala ValAsn Thr Ala Ala Gln Asn Asn Ala Asp Ala Leu PheVal Pro Pro Pro Asp Thr Pro Asn Ala Leu Ala TrpThr Ala Met Leu Asn Ala Gly Glu Ser Gly Ser ValThr Phe Arg Thr Pro Ala Pro Gly Thr Tyr Leu TyrIle Cys Thr Phe Pro Gly His Tyr Leu Ala Gly MetLys Gly Thr Leu Thr Val Thr Pro).SEQ ID NO: 17. Amino acid sequence of cucumberbasic protein from Cucumis sativus(Ala Val Tyr Val Val Gly Gly Ser Gly Gly Trp ThrPhe Asn Thr Glu Ser Trp Pro Lys Gly Lys Arg PheArg Ala Gly Asp Ile Leu Leu Phe Asn Tyr Asn ProSer Met His Asn Val Val Val Val Asn Gln Gly GlyPhe Ser Thr Cys Asn Thr Pro Ala Gly Ala Lys ValTyr Thr Ser Gly Arg Asp Gln Ile Lys Leu Pro LysGly Gln Ser Tyr Phe Ile Cys Asn Phe Pro Gly HisCys Gln Ser Gly Met Lys Ile Ala Val Asn Ala Leu).SEQ ID NO: 18. Amino acid sequence of Laz from Neisseria gonorrhoeae F62(Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu AlaThr Pro Ala Gly Glu Ala Pro Ala Ser Glu Ala ProAla Ala Glu Ala Ala Pro Ala Asp Ala Ala Glu AlaPro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu SerAsn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile GlnVal Ser Lys Ala Cys Lys Glu Phe Thr Ile Thr LeuLys His Thr Gly Thr Gln Pro Lys Ala Ser Met GlyHis Asn Leu Val Ile Ala Lys Ala Glu Asp Met AspGly Val Phe Lys Asp Gly Val Gly Ala Ala Asp ThrAsp Tyr Val Lys Pro Asp Asp Ala Arg Val Val AlaHis Thr Lys Leu Ile Gly Gly Gly Glu Glu Ser SerLeu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly AspTyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly AlaLeu Met Asn Gly Lys Val Thr Leu Val Asp).SEQ ID NO: 19. Amino acid sequence of the azurinfrom Vibrio parahaemolyticus(Met Ser Leu Arg Ile Leu Ala Ala Thr Leu Ala LeuAla Gly Leu Ser Phe Gly Ala Gln Ala Ser Ala GluCys Glu Val Ser Ile Asp Ala Asn Asp Met Met GlnPhe Ser Thr Lys Thr Leu Ser Val Pro Ala Thr CysLys Glu Val Thr Leu Thr Leu Asn His Thr Gly LysMet Pro Ala Gln Ser Met Gly His Asn Val Val IleAla Asp Thr Ala Asn Ile Gln Ala Val Gly Thr AspGly Met Ser Ala Gly Ala Asp Asn Ser Tyr Val LysPro Asp Asp Glu Arg Val Tyr Ala His Thr Lys ValVal Gly Gly Gly Glu Ser Thr Ser Ile Thr Phe SerThr Glu Lys Met Thr Ala Gly Gly Asp Tyr Ser PhePhe Cys Ser Phe Pro Gly His Trp Ala Ile Met GlnGly Lys Phe Glu Phe Lys),SEQ ID NO: 20. Amino acid sequence of amino acids57 to 89 of auracyanin B of Chloroflexus aurantiacus(His Asn Trp Val Leu Val Asn Gly Gly Asp Asp ValAla Ala Ala Val Asn Thr Ala Ala Gln Asn Asn AlaAsp Ala Leu Phe Val Pro Pro Pro Asp).SEQ ID NO: 21. Amino acid sequence of amino acids51-77 of Pseudomonas syringae azurin(Ser Lys Lys Ala Asp Ala Ser Ala Ile Thr Thr AspGly Met Ser Val Gly Ile Asp Lys Asp Tyr Val Lys Pro Asp Asp).SEQ ID NO: 22. Amino acid sequence of amino acids89-115 of Neisseria meningitidis Laz(Ile Gly Lys Thr Gln Asp Met Asp Gly Ile Phe LysAsp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp).SEQ ID NO: 23. Amino acid sequence of amino acids52-78 of Vibrio parahaemolyticus azurin(Ala Asp Thr Ala Asn Ile Gln Ala Val Gly Thr AspGly Met Ser Ala Gly Ala Asp Asn Ser Tyr Val Lys Pro Asp Asp).SEQ ID NO: 24. Amino acid sequence of amino acids51-77 of Bordetella bronchiseptica azurin(Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys AspGly Ile Ala Ala Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp).SEQ ID NO: 25. Amino acid sequence of p18, Pseudo-monas aeruginosa azurin residues 50-67(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val ThrAsp Gly Met Ala Ser Gly).SEQ ID NO: 26. Amino acid sequence of amino acids36-88 of Pseudomonas aeruginosa azurin(Pro Gly Asn Leu Pro Lys Asn Val Met Gly His AsnTrp Val Leu Ser Thr Ala Ala Asp Met Gln Gly ValVal Thr Asp Gly Met Ala Ser Gly Leu Asp Lys AspTyr Leu Lys Pro Asp Asp Ser Arg Val Ile Ala His Thr Lys Leu Ile Gly).SEQ ID NO: 27. Amino acid sequence of amino acids36 to 77 of Pseudomonas aeruginosa azurin(Pro Gly Asn Leu Pro Lys Asn Val Met Gly His AsnTrp Val Leu Ser Thr Ala Ala Asp Met Gln Gly ValVal Thr Asp Gly Met Ala Ser Gly Leu Asp Lys AspTyr Leu Lys Pro Asp Asp).SEQ ID NO: 28. Amino acid sequence of amino acids36 to 89 of Pseudomonas aeruginosa azurin(Pro Gly Asn Leu Pro Lys Asn Val Met Gly His AsnTrp Val Leu Ser Thr Ala Ala Asp Met Gln Gly ValVal Thr Asp Gly Met Ala Ser Gly Leu Asp Lys AspTyr Leu Lys Pro Asp Asp Ser Arg Val Ile Ala HisThr Lys Leu Ile Gly Ser).SEQ ID NO: 29. Amino acid sequence of amino acids36 to 128 of Pseudomonas aeruginosa azurin(Pro Gly Asn Leu Pro Lys Asn Val Met Gly His AsnTrp Val Leu Ser Thr Ala Ala Asp Met Gln Gly ValVal Thr Asp Gly Met Ala Ser Gly Leu Asp Lys AspTyr Leu Lys Pro Asp Asp Ser Arg Val Ile Ala HisThr Lys Leu Ile Gly Ser Gly Glu Lys Asp Ser ValThr Phe Asp Val Ser Lys Leu Lys Glu Gly Glu GlnTyr Met Phe Phe Cys Thr Phe Pro Gly His Ser AlaLeu Met Lys Gly Thr Leu Thr Leu Lys).SEQ ID NO: 30. Amino acid sequence of amino acids53 to 70 of Pseudomonas aeruginosa azurin(Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly MetAla Ser Gly Leu Asp Lys).SEQ ID NO: 31. Amino acid sequence of amino acids53 to 64 of Pseudomonas aeruginosa azurin(Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met).SEQ ID NO: 32. Amino acid sequence DGXXXXXDXXYXKXXD.SEQ ID NO: 33. Amino acid sequence DGXXXXDXXYXKXXD.SEQ ID NO: 34. Amino acid sequence of p18b,Pseudomonas aeruginosa azurin residues 60-77(Val Thr Asp Gly Met Ala Ser Gly Leu Asp Lys AspTyr Leu Lys Pro Asp Asp). SEQ ID NO: 35. Sequence of C-terminal 12 aminoacids of p28, Pseudomonas aeruginosa azurin residues 66-77 (p12)(Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 36. Sequence of C-terminal 10 aminoacids of p28, Pseudomonas aeruginosa azurin residues 68-77(Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 37. Sequence of C-terminal 11 aminoacids of p28, Pseudomonas aeruginosa azurin residues 67-77(Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 38 is the amino acid sequence of avariant of the azurin truncation p28(Leu Ser Thr Ala Ala Asp Met Gln Ala Val Val ThrAsp Thr Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 39 is the amino acid sequence of avariant of the azurin truncation p28(Leu Ser Thr Ala Ala Asp Leu Gln Gly Val Val ThrAsp Gly Leu Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 40 is the amino acid sequence of avariant of the azurin truncation p28(Leu Ser Thr Ala Ala Asp Val Gln Gly Val Val ThrAsp Gly Val Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 41 is the amino acid sequence of amodified cupredoxin derived peptide(Asp Asp Pro Lys Leu Tyr Asp Lys Asp Leu Gly SerAla Met Gly Asp Thr Val Val Gly Gln Met Asp Ala Ala Thr Ser Leu).SEQ ID NO: 42 is the amino acid sequence of amodified cupredoxin derived peptide(Acetylation-Leu Ser Thr Ala Ala Asp Met Gln GlyVal Val Thr Asp Gly Met Ala Ser Gly Leu Asp LysAsp Tyr Leu Lys Pro Asp Asp-amidation).SEQ ID NO: 43 is the amino acid sequence of a hexapeptide(Val Ser Pro Pro Ala Arg). SEQ ID NO: 44 is the amino acid sequence of ahexapeptide (Tyr Thr Pro Pro Ala Leu).SEQ ID NO: 45 is the amino acid sequence of a hexapeptide(Phe Ser Phe Phe Ala Phe). SEQ ID NO: 46 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Thr Pro Gly Cys).SEQ ID NO: 47 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Cys Gln Gly Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 48 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Cys Met Gln Gly Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 49 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Cys Asp Met Gln Gly Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 50 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Thr Met Gln Cys Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 51 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Thr Met Gln Gly Cys Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 52 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asn Thr Gln Gly Cys Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 53 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asn Thr Gln Gly Val Cys ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 54 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Thr Ala Val Cys ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 55 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Thr Ala Val Val CysAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 56 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Thr Val Val CysAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 57 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Thr Val Val ThrCys Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 58 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Ala Thr Val ThrCys Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 59 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Ala Thr Val ThrAsp Cys Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 60 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Thr AlaAsp Cys Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 61 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Thr AlaAsp Gly Cys Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 62 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val ThrAsn Gly Cys Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 63 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val ThrAla Thr Met Gly Ser Gly Leu Cys Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 64 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val ThrAsp Leu Thr Ala Ser Gly Leu Cys Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 65 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Trp Ala Ala Asp Met Gln Gly Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 66 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Trp Gly Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 67 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val TrpAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 68 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val ThrAsp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 69 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Trp Ala Ala Asp Met Trp Gly Val Val ThrAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 70 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Trp Ala Ala Asp Met Gln Gly Val Val TrpAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 71 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Trp Ala Ala Asp Met Gln Gly Val Val ThrAsp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 72 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Trp Gly Val Val TrpAsp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 73 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Trp Gly Val Val ThrAsp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 74 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val TrpAsp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 75 is the amino acid sequence of amodified cupredoxin-derived peptide(Leu Ser Trp Ala Ala Asp Met Trp Gly Val Val TrpAsp Trp Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp).SEQ ID NO: 76 is the amino acid sequence of amodified cupredoxin-derived peptide(X₁ Ser X₂ Ala Ala Asp X₃X₄X₅ Val Val X₆ AspX₇X₈ Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp X₉).SEQ ID NO: 77 is the amino acid sequence of amodified cupredoxin-derived peptide(X₁ Asp Pro Lys Leu Tyr Asp Lys Asp Leu Gly SerAla X₂X₃ Asp X₄ Val Val X₅X₆X₇ Asp Ala Ala X₈ Ser X₉).SEQ ID NO: 78 is a primer for pUC19-azu(5′-CGGGATCCCC GGCAACCTGC CGAAGAACGT CATGGGC-3′)SEQ ID NO: 79 is a primer for pUC19-azu(5′-CGGAATTCGC ATCACTTCAGG GTCAGGG-3′)SEQ ID NO: 80 is a primer for pGST-azu 36-50(5′-GGCCACAACT GGGTACTGTG AACCGCCGCC GACATGCAG-3′)SEQ ID NO: 81 is a primer for pGST-azu 36-50(5′-CTGCATGTCG GCGGCGGTTC ACAGTACCCA GTTGTGGCC-3′).SEQ ID NO: 82 is a primer for pGST-azu 36-77(5′-CCTGAAGCCC GACGACTGAC GTGTCATCGC CCACACC-3′)SEQ ID NO: 83 is a primer for pGST-azu 36-77(5′-GGTGTGGGCG ATGACACGTC AGTCGTCGGG CTTCAGG-3′).SEQ ID NO: 84 is a primer for pGST-azu 36-89(5′-CCAAGCTGAT CGGCTCGTGA GAGAAGGACT CGGTGACC-3′).SEQ ID NO: 85 is a primer for pGST-azu 36-89(5′-GGTCACCGAG TCCTTCTCTC ACGAGCCGAT CAGCTTGG-3′).SEQ ID NO: 86 is a primer for azu 50-77(5′-CGGGATCCTG AGCACCGCCG CCGACATGCA GGG-3′).SEQ ID NO: 87 is a primer for an 67-77(5′-CGGGATCCCC GGCCTGGAC AGGATTACCT GAAGCCCG-3′)SEQ ID NO: 88 is a reverse primer(5′-CGGAATTCGC ATCACTTCAG GGTCAGGG-3′).SEQ ID NO: 89 is a primer for pGST-azu-50-66(5′-GACGGCATGG CTTCCTGACT GGACAAGGAT TACC-3′)SEQ ID NO: 90 is a primer for pGST-azu-50-66(5′-GGTAATCCTT GTCCAGTCAG GAAGCCATGC CGTC-3′).SEQ ID NO: 91 is a forward primer (5′-CGGGATCCCC ATGGTGAGCA AGGGCG-3′)SEQ ID NO: 92 is a reverse primer(5′-CGGAATTCCT TGTACAGCTC GTCCATGCCG-3′)SEQ ID NO: 93 is a primer for pGST-azu 50-77(5′-CCGCTCGAGC CTGAGCACCG CCGCCATGCA GGG-3′)SEQ ID NO: 94 is a primer for pGST-azu 50-77(5′-TTTTCCTTTT GCGGCCGCTC AGTCGTCGGG CTTCAGGTAA TC C-3′).SEQ ID NO: 95 is the amino acid sequence of polyarginine, or Arg8 (Arg Arg Arg Arg Arg Arg Arg Arg)SEQ ID NO: 96 is the amino acid sequence of a sectionof p18 (Ser Gly Leu Asp Lys Asp).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts photographs of all of the glands evaluated for theefficacy of p28 and azurin. FIG. 1A shows a representative photograph ofalveolar lesions in a DMBA-treated gland and its comparison with a glandthat was treated with DMBA along with a chemopreventive agent. FIGS.1B-1G show representative photographs of the effects of p28 on thedevelopment of alveolar lesions.

FIG. 2 depicts a graph showing the efficacy of p28 against DMBA-inducedmammary alveolar lesions.

FIG. 3 depicts photographs of representative sections of ductal lesionsand effect of p28.

FIG. 4 depicts a graph showing the efficacy of p28 against DMBA-inducedductal lesions

FIG. 5. Diagram showing the localization of the α-helix in wt-azurin aswell as in the wt-azurin 50-77 protein transduction domain. Replacementof three amino acids in the azurin 50-77 domain by proline residues isindicated.

FIGS. 6(A), (B) and (C). (A) Diagram showing construction of aGST-GFP-azu 50-77 fusion protein. The gfp gene was introduced at the3′-end of the gst gene (for GST-GFP) and the azu 50-77 fragment was thenligated at the 3′-end of the gfp gene in frame to produce theGST-GFP-azu 50-77 fusion protein. GST-GFP-azu 50-77 was purified as asingle fusion protein from the cell lysates. Purified proteins were runon SDS-PAGE and detected by Coomassie Blue staining (6(B) and also byWestern blotting using anti-azurin antibody (6(C)).

FIGS. 7(A), (B) and (C). Diagrams showing a kinetic study for theinternalization of GST-Green Fluorescent Protein (GFP) andGST-GFP-azurin fusion proteins. Green fluorescence was assayed in J774cells treated with various concentrations of GST-GFP (10(a)) orGST-GFP-azu 50-77 (10(b)) at 37° C. for 1 hr. Ten thousand cells wereanalyzed by flow cytometry. (c) Time-dependence of internalization ofGST-GFP-azu 50-77. J774 cells were incubated with 200 μg/ml GST-GFP-azu50-77 for indicated times at 37° C. and analyzed by flow cytometry.

FIGS. 8(A), (B) and (C). (A) Diagram showing the exotoxin A domain III(amino acids 405-613), as well as part of domain 1b (amino acids381-404), fused to GST (GST-PEDIII) as earlier described for the GST-GFPfusion. The azu 50-77 fragment was then ligated to the carboxyl end ofGST-PEDIII (GST-PEDIII-azu 50-77), using PCR. (B) The fusion proteinswere purified by glutathione Sepharose 4B column gel filtration columnchromatography and run on SDS-PAGE for size determination. (C) Diagramshowing action of GST-PEDIII-azu 50-77 fusion protein in UISO-Mel-2cancer cells and in normal fibroblast (FBT) cells, as determined byPEDIII-mediated cytotoxicity. Various concentrations, as indicated, ofGST-PEDIII and GST-PEDIII-azu 50-77 were incubated with UISO-Mel-2 andFBT cells for 24 h, after which the cell viability was determined by MTTassay.

FIG. 9. Diagram showing PEDIII-mediated cytotoxicity ofGST-PEDIII-rusticyanin fusion protein against UISO-Mel-2 cancer cellsand FBT cells. Various concentrations, as indicated, of GST-PEDIII andGST-PEDIII-azu 50-77 were incubated with UISO-Mel-2 and FBT cells for 24h, after which the cell viability was determined by MTT assay.

FIGS. 10, (A), (B) (C) and (D). Depicts photographs showing penetrationof azurin derived peptides, p18 and p28, into cancer cell lines ofdiverse histogenesis and their normal counterparts. (A) and (B) Photosshowing penetration of Alexafluor 568 labeled p28 or p18 after 2 hrs at37° C. The cationic Args was used as a control. (C) Graphs depictingflow cytometric analysis of the penetration of Alexafluor 568 labeledp28 or p18 into the same cell lines after 2 hrs at 37° C. (D) Graphsdepicting fold increase over fluorescence from normal cells. Similarobservations of p28 or p18 entry into 4 melanoma cell lines show aseveral fold increase over fluorescence from normal cells.

FIG. 11, (A) and (B). Depicts photographs showing entry of azu 60-77(p18b) and azu 66-77 (p12) into cancer and normal cells. Cells wereincubated with alexafluor 568 labeled p18b (A) or p12 (B) at 37° C. for2 hrs and images recorded by confocal microscopy.

FIG. 12, (A) and (B). Graphs depicting cellular membrane toxicity ofazurin and its peptides. (A) LDH leakage assay of UISOMel-2 cellsexposure for 10 min to different concentrations of p28, p18 and azurinat 37° C. A standard lysis buffer (cytotox-one reagent) was included asa positive control. Changes in fluorescence following exposure weremeasured at λ_(ex) 560 nm and λ_(em) 590 nm. Lysis buffer was defined as100% LDH release. Data represent % of positive fluorescence of control.Data are shown as mean±SEM. (B) Hemoglobin leakage from humanerythrocytes incubated with p28, p18 and azurin. Human erythrocytes wereincubated with peptide for 30 min at 37° C. and absorbance at 540 nmdetermined. Hemoglobin release following 0.1% Triton X-100 was definedas 100% hemoglobin release. Data represent mean±SEM of triplicatedeterminations.

FIG. 13, (A), (B), (C) and (D). Depicts photographs showing temperaturedependent and competitive internalization of p28 and p18 into UISO-Mel-2cells. Penetration of Alexafluor 568 labeled p28 (A) or p18 (B) at 2011Mwas evaluated by confocal microscopy at different temperatures. (C) and(D) Confocal analysis of entry of Alexafluor 568 labeled p28 (C) or p18(D) at 5 μM into UISO-Mel-2 cells after 30 min at 37° C. in thepresence/absence of unlabeled peptide (200 fold excess).

FIG. 14, (A), (B), (C) and (D). (A) Depicts photographs showing confocalanalysis of 28, p18 (20 μM) and Arg₈ (SEQ ID NO: 95) (10 μM) entry intoUISO-Mel-2 cells after 1 hr at 37° C. in the presence/absence of heparinsulfate (100 μg/ml). (B) Graphs showing flow cytometric analysis of p28or p18 entry in the presence of inhibitors. Cell fluorescence intensityin the absence of inhibitor (control) was considered as 100%. (C) Graphsdepicting FRCS analysis of p28 and p18 entry into fibroblasts inpresence of inhibitors. (D) Depicts photographs showing colocalizationof p18 and p28 with caveolin I (Panel 1). UISO-Mel-2 cells wereincubated with Alexafluor 568 labeled p18 or p28 (20 μM) or media for 2hrs at 37° C. Cells were fixed and processed for anti-caveolin 1immunostaining. Confocal analysis of entry of Alexafluor 568 labeled p18or p28 (20 μM) into UISO-Mel-2 cells after 2 hrs at 37° C. followed byantigolgin 97 antibodies (Panel 2). Colocalization of Alexafluor 568labeled azurin, p28 and p18 (red) with mitotracker (green) (Panel 3) andLysotracker (green) (Panel 4) dyes in UISO-Mel-2 cells. Cells wereincubated at 37° C. with 20 μM azurin, p28, p18 or media only. After 90min incubation, mitotracker/lysotracker probes were added and cellsincubated for 30 min. Cells were counterstained with DAPI (blue).Colocalization of azurin, p28 or p18 appears as a yellow florescence.

FIG. 15, (A) and (B). Graphs depicting UISO-Mel-2 cells that wereincubated with increasing concentrations of azurin, p28, or p18 at 37°C. for 72 hrs. MTT (A); Direct cell count (B). Cell viability (MTT) orcell number in control wells were considered as 100%. Data representmean±SEM.

FIG. 16, (A) through (H). Depict photographs showing uptake of compoundsby cells, taken using a confocal microscope after treatment of cellswith proteins and/or buffer. (A) Human brain tumor LN-229 cells werepretreated with 20 μM of unlabeled proteins or PBS buffer for 2 hours,then washed three times using PBS buffer. All buffer was discarded andthen 20 μM of Alex568-Paz was added for 30 minutes at 37° C. (B) TheLN-229 cells were then treated with 20 μM of unlabeled proteins or PBSbuffer and 20 μM of Alex568-Paz for 30 minutes at 37° C. (C) Anothergroup of human brain tumor LN-229 cells were pretreated with 10 μMunlabeled proteins or PBS buffer for 2 hours, then washed three timesusing PBS buffer. All buffer was discarded and then 10 μM of Alex568-Pazwas added for 30 minutes at 37° C. (D) The LN-229 cells were thentreated with 10 μM unlabeled proteins or PBS buffer and 10 μM ofAlex568-Paz for 30 minutes at 37° C. (E) Human brain tumor LN-229 cellswere treated with 20 μM of unlabeled proteins or PBS buffer and 20 μM ofAlex568-Paz for 30 minutes at 37° C. (F) Human brain tumor LN-229 cellswere treated with 20 μM of Alex568-H.8 for 30 minutes at 37° C. (G)Human brain tumor LN-229 cells were treated with 20 μM Alex568-proteinsfor 30 minutes at 37° C. (H) Human breast adenocarcinoma MCF-7 cellswere treated with 20 μM of Alex568-proteins for 30 minutes at 37° C.

FIG. 17, (A) through (C). Graphs and charts depicting peptide bindingand entry into cells. (A) UISO-Mel-2 or fibroblast cells (3×10⁵ cells)were suspended in MEME media without phenol red. Reactions were startedby adding Alexafluor 568-conjugated p28 at 10, 50, 100, 150, 250, 300and 400 μM for 30, 60, 90 and 120 see on ice. Cells were analyzed byflow cytometry. (B) The K_(m) and V_(max) were calculated by plottingpeptide concentration (μM) vs velocity (MFI/sec). (C) Peptide bindingand entry was determined using whole Mel2 cells (50,000 cells/ml), wereincubated for 30 min at 37° C. with increasing concentrations (0-175 nM)of radiolabeled azurin in the presence/absence of 1000 fold excess ofunlabeled p28, or azurin, and radioactivity remaining in the cell pelletcounted using a gamma counter. Radioactivity in cells incubated with¹²⁵I azurin alone was considered total binding; radioactivity in thepresence of unlabeled azurin or p28 was considered nonspecific binding.Specific binding was determined by subtracting nonspecific binding fromtotal binding and Scatchard plots generated.

FIG. 18, (A) through (C). Depict side and back photographs of mice withmelanoma MEL-23 tumors taken after injection with p28 dye complex at 60μmolar concentration in 250 μL scans and after injection with controlPBS at (A) 24 hours and (B) 48 hours. (C) depics side and backphotographs of mice with melanoma MEL-23 tumors taken after injectionwith p28 at 200 μM concentration at 24 and 48 hours.

FIG. 19, (A) through (C). Depict side and back photographs of mice withmelanoma MEL-23 tumors taken after injection with p18 at 60 μmolarconcentration at (A) 17 hours, (B) 24 hours, and (C) 46 hours. (C) alsodepicts photographs of mouse organs, including the heart, lung, liver,kidney, spleen, and brain, taken 46 hours after injection of p18.

FIG. 20, (A) and (B). (A) Depicts side and back photographs of mice withtumors taken 12 hours after injection with p18, p28, and arg-8 (SEQ IDNO: 95) at 60 μmolar concentration. (B) Depicts photographs of mouseorgans, including mouse brains, taken 12 hours after injection with p18,p28, and arg-8 (SEQ ID NO:95).

FIG. 21, (A) and (B). (A) Depicts side and back photographs of mice withmelanoma MEL-6 tumors taken 40 hours after injections of 600 μMconcentrations of p18 and arg-8 (SEQ ID NO: 95) into tail veins. Animalstreated with p18 received 0.5 million cells, and animals treated witharg-8 (SEQ ID NO: 95) received 1 million cells. (B) Depicts photographsof mouse organs taken 40 hours after injections of 600 μM concentrationsof p18 and arg-8 (SEQ ID NO: 95).

FIG. 22, (A) and (B). (A) Depicts side and back photographs of mice withmelanoma MEL-23 tumors taken 16 hours after injections of 60 μMconcentrations of p28, p18, and arg-8 (SEQ ID NO: 95). (B) Depicts sideand back photographs of mice with melanoma MEL-23 tumors taken 24 hoursafter injections of 60 μM concentrations of p28, p18, and arg-8 (SEQ IDNO: 95).

FIG. 23. Depicts photographs of mouse organs taken 48 hours afterinjection of 60 μM concentrations of p28 and p18 dye peptide complexinto mice with melanoma MEL-23.

FIG. 24. Depicts photographs of mouse organs taken 24 hours afterinjection of 60 μM concentrations of p28 into mice with MEL-23 tumorsand organs.

FIG. 25. Depicts side and back photographs of mice with melanoma MEL-23tumors taken 16 hours after injections of 60 μM concentrations of p28and arg-8 (SEQ ID NO: 95).

FIG. 26. Depicts side and back photographs of mice with melanoma MEL-23tumors taken 16 hours after injections of 60 μM concentrations of p18.

FIG. 27. Depicts side photographs of mice with tumors taken 10 and 24hours after high dose treatment with 240 μM concentrations of p18, p28,and arg-8 (SEQ ID NO: 95).

FIG. 28. Depicts side and back photographs of mice with MCF-7 tumors andorgans taken 28 hours after high dose treatment with 240 μMconcentrations of p18, p28, and arg-8 (SEQ ID NO: 95). Also depictsphotographs of mouse organs with MCF-7 taken 28 hours after high dosetreatment with 240 μM concentrations of p18, p28, and arg-8 (SEQ ID NO:95).

FIG. 29. Depicts side and back photographs of mice with tumors taken 50hours after high dose treatment with 240 μM concentrations of p18, p28,and arg-8 (SEQ ID NO: 95).

FIG. 30. Depicts photographs of mouse organs taken 24 hours afterinjection of 120 μM concentrations of p18, p28, and arg-8 (SEQ ID NO:95) into the tail veins of mice with HCT-116 tumors and organs.

FIG. 31, (A) and (B). (A) Depicts photographs of mouse organs taken 24hours after injection of 120 μM concentrations of p18, p28, and arg-8(SEQ ID NO: 95) into the tail veins of mice with HCT-116 tumors andorgans. (B) Depicts side photographs of mice with HCT-116 tumors taken21 hours after injection of 120 μM concentrations of p18, p28, and arg-8(SEQ ID NO: 95) into their tail veins.

FIG. 32, (A) and (B). (A) Depicts side and back photographs of mice withHCT-116 24 hours after injection with 120 μM concentrations of p28, 47days after injection of 1 million cells into tail veins. (B) Depictsphotographs of mouse organs taken from mice with HCT-116 4 hours afterinjection with 120 μM concentrations of p28, 47 days after injection of1 million cells into tail veins.

FIG. 33. Depicts photographs of organs from MEL-6 mice taken 24 hoursafter treatment with 120 μM concentrations of p18, p28, and arg-8 (SEQID NO: 95).

FIG. 34, (A) and (B). (A) Depicts side and back photographs of MEL-6mice taken 22 hours after injection of 120 μM concentrations of p18,p28, and arg-8 (SEQ ID NO: 95), and 60 60 μM concentration of arg-8 (SEQID NO: 95). (B) Depicts photographs of MEL-6 mouse organs aftertreatment with 120 μM concentrations of p18, p28, and arg-8 (SEQ ID NO:95), and 60 μM concentration of arg-8 (SEQ ID NO: 95).

FIG. 35, (A) and (B). (A) Depicts photographs of organs from HT-1080mice taken 22 hours after treatment with 60 and 120 μM concentrations ofp18, p28, and arg-8 (SEQ ID NO: 95). (B) Depicts side-by-sidephotographs of brains from HT-1080 mice taken 22 hours after treatmentwith 60 and 120 μM concentrations of p18, p28, and arg-8 (SEQ ID NO:95), demonstrating the differences between uptake of p18 and p28 intothe brain.

FIG. 36. Depicts side and back photographs of HT-1080 mice duringDoxorubicin vs. p28 study taken 16 hours after treatment with 60 and 120μM concentrations of p18, p28, and arg-8 (SEQ ID NO: 95).

FIG. 37, (A) and (B). (A) Depicts photographs of organs from HT-1080mice taken 22 hours after treatment with 60 and 120 μM concentrations ofp28 and arg-8 (SEQ ID NO: 95). (B) Depicts side-by-side photographs ofbrains from HT-1080 mice taken 22 hours after treatment with 60 and 120μM concentrations of p28 and arg-8 (SEQ ID NO: 95).

FIG. 38, (A) and (B). (A) Depicts photographs of organs from HT-1080mice taken 22 hours after treatment with 60 and 120 μM concentrations ofp18 and arg-8 (SEQ ID NO: 95). (B) Depicts side-by-side photographs ofbrains from HT-1080 mice taken 22 hours after treatment with 60 and 120μM concentrations of p18 and arg-8 (SEQ ID NO: 95).

FIG. 39, (A) through (E). Depicts photographs of HT-1080 mice with lungmetastases treated via their tail veins with (A) 3 mg/kg Doxorubicin IP,3 treatments; (B) 5 mg/kg IP p28 daily; (C) PBS control, PBS IP daily;(D) 10 mg/kg IP p28 daily; (E) 20 mg/kg IP daily.

FIG. 40, (A) and (B). (A) Depicts photographs of organs from HT-1080mice in an animal study, whereby 1×10⁶ cells are injected into tailveins (43 days) and all treated mice have lung metastases, taken 24 and26 hours after 60 μM concentrations of p28 injected into tail veins.Animal 6982 was dead when photographed. (B) Depicts side and backphotographs of HT-1080 mice in an animal study, whereby 1×10⁶ cells areinjected into tail veins (43 days), taken 22 hours after 60 μMconcentrations of p28 injected into tail veins. Animal 6982 was deadwhen photographed.

FIG. 41. Depicts side and back photographs of HT-1080 mice in an animalstudy, whereby 1×10⁶ cells are injected into tail veins (43 days), taken26 hours after 60 μM concentrations of p28 injected into tail veins.

FIG. 42, (A) and (B). Depicts photographs of (A) organs from mice and(B) back views of mice in Balb-C peptide study taken 12 hours aftertreatment with 60 and 120 μM concentrations of p18, p28, and arg-8 (SEQID NO: 95).

FIG. 43, (A) and (B). Depicts photographs of (A) organs from mice and(B) side views of mice in Balb-C peptide study taken 24 hours aftertreatment with 60 and 120 μM concentrations of p18, p28, and arg-8 (SEQID NO: 95).

FIG. 44. Depicts side and back photographs of MEL-6 mice (0.5 millioncells injected via tail vein) 16 hours after injection into tail veinsof 60 μM concentrations of p18 and arg-8 (SEQ ID NO: 95).

FIG. 45, (A) through (D). Depicts photographs of mouse organs, andspecifically mouse brains, after treatment with p18 and p28.

FIG. 46. Depicts photographs of organs from MEL-6 mice taken 24 hoursafter treatment with p28, p18, and arg-8 (SEQ ID NO: 95).

FIG. 47, (A) through (C). (A) Depicts side and back photographs of MEL-6mice 3 hours after injection with 60 μM concentrations of p18, p28, andarg-8 (SEQ ID NO: 95). (B) Depicts side and back photographs of MEL-6mice, and photographs of organs from MEL-6 mice, taken 22 hours afterinjection with 60 μM concentrations of p18, p28, and arg-8 (SEQ ID NO:95). (C) Depicts photographs of organs from MEL-6 mice 24 hours afterinjection with 60 μM concentrations of p18, p28, and arg-8 (SEQ ID NO:95).

FIG. 48, (A) and (B). Depict uptake of p18 and p28 into (A) mouse brainsand (B) mouse organs).

FIG. 49. Depicts side and back photographs of MEL-6 mice in studywhereby 0.5 million cells injected I.V. into tail vein (44 days post),taken 120 hours after injection into tail vein of 24 μM concentrationsof p18 and arg-8 (SEQ ID NO: 95).

FIG. 50. Depicts photographs of organs from MEL-6 mice taken 168 hoursafter treatment with p18.

FIG. 51. Depicts side and back photographs of MEL-6 mice taken 72 hrsafter injection of arg-8 (SEQ ID NO: 95) and p18, 41 days post injectionof cells.

FIG. 52. Depicts back photographs of mice taken after injection of arg-8(SEQ ID NO: 95) and p18.

FIG. 53. Depicts side and front photographs of mice taken 3, 24, and 48hours after injection of arg-8 (SEQ ID NO: 95) and p18.

FIG. 54. Graphs depicting growth inhibition of human breast cancer cellsby p28. MCF-7 cells were incubated with p28 (0-200 μM) at 37° C. for 24,48 and 72 h. Cell count (A) and MTT assays (B). Doxorubicin (10 μM) wasused as a positive control. Cell number or viability of control wellswere considered as 100%. Data represent mean % of control±SEM. *,p<0.05. (C) Inhibition of MCF-7 xenograft growth by p28. A minimum of 10mice per group were treated with paclitaxel 15 μmol/kg i.p. on days 10,14, 21 and 25 or 5 or 10 mg/kg p28 i.p. daily for 30 days. Barsrepresent Mean±SEM. *, p<0.05.

FIG. 55. (A) and (B) are graphs depicting FACS analyses of cell cycleand penetration of breast cancer cells by p28. MCF-7 (A) and MDD2 cells(B) were treated with p28 (50 μM) for 48 and 72 hr. Cells were stainedwith propidium iodide and analyzed by flow cytometry as described inYamada, et al., Proc Natl Acad Sci USA, 101:4770-4775 (2004). Thepercentage of cells in the G₁, S, G₂/M and sub-G₁ (apoptosis) phases areindicated. (C) contains photographs depicting MCF-7 and MDD2 cellscultured on cover slips overnight in phenol-red free MEM, which weretreated with 20 μM p28 or 10 μM of the cationic (positive control)peptide, octaarginine (Arg₈) (SEQ ID NO: 95), for 2 hr at 37° C.Red-Alexa fluor 56

FIG. 56. Interaction of p28 with p53. (A)-(D) are photographs depictingp53 and p28 levels in cells. (A) p53 levels in MCF-7 cells with timeafter incubation with p28. (%) increase relative to p53 levelimmediately prior to treatment (0 hr as 100%). (B) GST pull-down assaydemonstrating complex formation between GST-p28 and p53. Left to rightGST-p28 (10 and 20 μg/reaction), GST-MDM2 and GST alone. p53 wasdetected by immunoblotting (IB) using anti-p53 antibody. (C) p53 waspulled down by GST-MDM2 in the presence of a molar excess of p28(upper). Three different anti-p53 antibodies, Pab 1801 (32-79 aminoacids), ab 2433 (277-296 amino acids) and Pab1802 (306-393 amino acids)reacted with GST-p53 immobilized beads in the presence of p28. p28detected by IB using an anti-p28 antibody (lower). (D) Competition forp28 binding to GST-p53 by a molar excess of p28 fragments p12, p18 andp18b. Relative amount of binding (p28 alone expressed as 100%). M: p28marker. (E) is a graph depicting p53 DNA-binding in MCF-7 nuclearextracts after exposure to p28 or azurin. Nuclear extracts ofH₂O₂-treated MCF-7 cells served as an internal control. Thep53-oligonucleotide complex was quantified with a monoclonal antibody top53. Data are expressed as Mean±SEM of triplicates.

FIG. 57. Photographs depicting induction of the cyclin (CDK and CDKI)cascade by p28. MCF-7 (A) and MDD2 cells (B) were exposed to p28 (50 μM)for 24, 48 and 72 hr and protein levels determined by immunoblotting.Intracellular localization and relative level of p21 (C) and cyclin B1(D) MCF-7 cells were cultured on cover slips with p28 for 72 h. p21 andcyclin B were stained with corresponding to the specific antibodies. (E)Phosphorylated cdc2 was estimated with an anti p-cdc2 antibody (SantaCruz Biotechnology, CA). All results normalized by actin as an internalcontrol

FIG. 58. Pictures depicting protein structures. A) Azurin truncationwith alpha-helical structure; B) Result of 70 ns simulation; C)Measurement of thioether bridge positions based on distances between Cαatoms in a simulated structure.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “cell” includes either the singular or theplural of the term, unless specifically described as a “single cell.”

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid. The terms also apply to naturally occurring aminoacid polymers. The terms “polypeptide,” “peptide,” and “protein” arealso inclusive of modifications including, but not limited to,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation. It will beappreciated that polypeptides are not always entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination andthey may be circular (with or without branching), generally as a resultof post-translation events, including natural processing event andevents brought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods aswell.

As used herein, the term “pharmacologic activity” means the effect of adrug or other chemical on a biological system. The effect of chemicalmay be beneficial (therapeutic) or harmful (toxic). The pure chemicalsor mixtures may be of natural origin (plant, animal, or mineral) or maybe synthetic compounds.

As used herein, the term “premalignant” means precancerous, or beforeabnormal cells divide without control.

As used herein, the term “lesion” means an area of abnormal tissue.

As used herein, the term “pathological condition” includes anatomic andphysiological deviations from the normal that constitute an impairmentof the normal state of the living animal or one of its parts, thatinterrupts or modifies the performance of the bodily functions, and is aresponse to various factors (as malnutrition, industrial hazards, orclimate), to specific infective agents (as worms, parasitic protozoa,bacteria, or viruses), to inherent defects of the organism (as geneticanomalies), or to combinations of these factors.

As used herein, the term “condition” includes anatomic and physiologicaldeviations from the normal that constitute an impairment of the normalstate of the living animal or one of its parts, that interrupts ormodifies the performance of the bodily functions.

As used herein, the term “suffering from” includes presently exhibitingthe symptoms of a pathological condition, having a pathologicalcondition even without observable symptoms, in recovery from apathological condition, or recovered from a pathological condition.

As used herein, the term “chemoprevention” is the use of drugs,vitamins, or other natural or synthetic agents, which may be biologic orchemical, to try to reduce the risk of, prevent, suppress, reverse, ordelay the development, or recurrence of, cancer.

As used herein, the term “cytotoxic” refers to the quality of beingtoxic to cells. For example, a “cytotoxic cupredoxin” is a cupredoxin orvariant, derivative, truncation, or structural equivalent thereof thatis toxic to cells, including cancer cells.

A used herein, the term “treatment” includes preventing, lowering,stopping, or reversing the progression or severity of the condition orsymptoms associated with a condition being treated. As such, the term“treatment” includes medical, therapeutic, and/or prophylacticadministration, as appropriate. Treatment may also include preventing orlessening the development of a condition, such as cancer.

As used herein, the term “inhibit cell growth” means the slowing orceasing of cell division and/or cell expansion. This term also includesthe inhibition of cell development or increases in cell death.

A “therapeutically effective amount” is an amount effective to prevent,lower, stop or reverse the development of, or to partially or totallyalleviate the existing symptoms of a particular condition for which thesubject being treated. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art.

The term “substantially pure,” as used herein, when used to modify aprotein or other cellular product of the invention, refers to, forexample, a protein isolated from the growth medium or cellular contents,in a form substantially free of, or unadulterated by, other proteinsand/or other compounds. The term “substantially pure” refers to a factorin an amount of at least about 75%, by dry weight, of isolated fraction,or at least “75% substantially pure.” More specifically, the term“substantially pure” refers to a compound of at least about 85%, by dryweight, of isolated fraction, or at least “85% substantially pure.” Mostspecifically, the term “substantially pure” refers to a compound of atleast about 95%, by dry weight, of isolated fraction, or at least “95%substantially pure.” The term “substantially pure” may also be used tomodify a synthetically-made protein or compound of the invention, where,for example, the synthetic protein is isolated from the reagents andby-products of the synthesis reaction(s).

The term “pharmaceutical grade,” as used herein, when referring to apeptide or compound of the invention, is a peptide or compound that isisolated substantially or essentially from components which normallyaccompany the material as it is found in its natural state, includingsynthesis reagents and by-products, and substantially or essentiallyisolated from components that would impair its use as a pharmaceutical.For example, a “pharmaceutical grade” peptide may be isolated from anycarcinogen. In some instances, “pharmaceutical grade” may be modified bythe intended method of administration, such as “intravenouspharmaceutical grade,” in order to specify a peptide or compound that issubstantially or essentially isolated from any substance that wouldrender the composition unsuitable for intravenous administration to apatient. For example, an “intravenous pharmaceutical grade” peptide maybe isolated from detergents, such as SDS, and anti-bacterial agents,such as azide.

The terms “isolated,” “purified” or “biologically pure” refer tomaterial which is substantially or essentially free from componentswhich normally accompany the material as it is found in its nativestate. Thus, isolated peptides in accordance with the inventionpreferably do not contain materials normally associated with thepeptides in their in situ environment. An “isolated” region of apolypeptide refers to a region that does not include the whole sequenceof the polypeptide from which the region was derived. An “isolated”nucleic acid, protein, or respective fragment thereof has beensubstantially removed from its in vivo environment so that it may bemanipulated by the skilled artisan, such as but not limited to,nucleotide sequencing, restriction digestion, site-directed mutagenesis,and subcloning into expression vectors for a nucleic acid fragment aswell as obtaining the protein or protein fragment in substantially purequantities.

The term “variant” as used herein with respect to a peptide, refers toamino acid sequence variants which may have amino acids replaced,deleted, or inserted as compared to the wild-type polypeptide. Variantsmay be truncations of the wild-type peptide. A “deletion” is the removalof one or more amino acids from within the polypeptide, while a“truncation” is the removal of one or more amino acids from one or bothends of the polypeptide. Thus, a variant peptide may be made bymanipulation of genes encoding the polypeptide. A variant may be made byaltering the basic composition or characteristics of the polypeptide,but not at least some of its pharmacologic activities. For example, a“variant” of azurin can be a mutated azurin that retains its ability toinhibit the development of premalignant mammalian cells. In some cases,a variant peptide is synthesized with non-natural amino acids, such asε-(3,5-dinitrobenzoyl)-Lys residues. Ghadiri & Fernholz, J. Am. Chem.Soc., 112:9633-9635 (1990). In some embodiments, the variant has notmore than 20 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 15 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 10 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 6 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 5 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 3 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In other embodiments, the variant iscreated using the methods and techniques disclosed herein.

The term “amino acid,” as used herein, means an amino acid moiety thatcomprises any naturally-occurring or non-naturally occurring orsynthetic amino acid residue, i.e., any moiety comprising at least onecarboxyl and at least one amino residue directly linked by one, twothree or more carbon atoms, typically one (α) carbon atom.

The term “derivative” as used herein with respect to a peptide refers toa peptide that is derived from the subject peptide. A derivationincludes chemical modifications of the peptide such that the peptidestill retains some of its fundamental activities. For example, a“derivative” of azurin can, for example, be a chemically modified azarinthat retains its ability to inhibit angiogenesis in mammalian cells.Chemical modifications of interest include, but are not limited to,amidation, acetylation, sulfation, polyethylene glycol (PEG)modification, phosphorylation or glycosylation of the peptide, or othermethods and techniques disclosed herein. In addition, a derivativepeptide may be a fusion of a polypeptide or fragment thereof to achemical compound, such as but not limited to, another peptide, drugmolecule or other therapeutic or pharmaceutical agent or a detectableprobe.

The term “percent (%) amino acid sequence identity” is defined as thepercentage of amino acid residues in a polypeptide that are identicalwith amino acid residues in a candidate sequence when the two sequencesare aligned. To determine % amino acid identity, sequences are alignedand if necessary, gaps are introduced to achieve the maximum % sequenceidentity; conservative substitutions are not considered as part of thesequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Oftenpublicly available computer software such as BLAST, BLAST2, ALIGN2 orMegalign (DNASTAR) software is used to align peptide sequences. In aspecific embodiment, Blastp (available from the National Center forBiotechnology Information, Bethesda Md.) is used using the defaultparameters of long complexity filter, expect 10, word size 3, existence11 and extension 1.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=X/Y*100

where

X is the number of amino acid residues scored as identical matches bythe sequence alignment program's or algorithm's alignment of A and B and

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A. When comparinglonger sequences to shorter sequences, the shorter sequence will be the“B” sequence. For example, when comparing truncated peptides to thecorresponding wild-type polypeptide, the truncated peptide will be the“B” sequence.

General

In some embodiments, the present invention provides compositionscomprising cupredoxin, and variants, derivatives, truncations, andstructural equivalents of cupredoxins, and methods to prevent thedevelopment of cancer in mammals. In other embodiments, the presentinvention provides compositions comprising cupredoxin, and variants,derivatives, truncations, and structural equivalents of cupredoxins thatpreferentially enter mammalian cells, including cancer cells. Otherembodiments provide variants, derivatives and structural equivalents ofcupredoxin that retain the ability to prevent the development of canceror the re-occurrence of cancer in mammals. Particular embodimentsprovide compositions comprising Pseudomonas aeruginosa azurin, variants,derivatives and structural equivalents of azurin, and their use to treatpatients, and particularly patients at a higher risk of developingcancer than the general population.

Other embodiments of the invention include methods to directly and/orpreferentially penetrate cancer cells. Further embodiments of theinvention include methods to directly and/or preferentially penetratecancer and normal cells and have chemopreventive effects therein. Otherembodiments provide methods to preferentially deliver therapeuticcompounds to cancer cells. Finally, the invention provides methods tostudy the development of cancer in mammalian cells, tissues and animalsby contacting the cells with a cupredoxin, or variant, derivative orstructural equivalent thereof, before or after inducing premalignantlesions, and observing the development of premalignant and/or malignantcells. Yet other embodiments will be evident from the disclosure herein.

Preferential Entry Into Cells

Previously, it was known that a redox protein elaborated by Pseudomonasaerugisnosa, the cupredoxin azurin, selectively enters J774 lung cancercells but not normal cells, and induces apoptosis. Zaborina et al.,Microbiology 146:2521-2530 (2000). Azurin can also selectively enter andkill human melanoma UISO-Mel-2 or human breast cancer MCF-7 cells.Yamada et al., PNAS 99:14098-14103 (2002); Punj et al., Oncogene23:2367-2378 (2004). Azurin from P. aeruginosa preferentially entersJ774 murine reticulum cell sarcoma cells, forms a complex with andstabilizes the tumor suppressor protein p53, enhances the intracellularconcentration of p53, and induces apoptosis. Yamada et al., Infectionand Immunity 70:7054-7062 (2002). Detailed studies of various domains ofthe azurin molecule showed that amino acids 50-77 (p28) (SEQ ID NO: 2)represented a protein transduction domain PTD) critical forinternalization and subsequent apoptotic activity. Yamada et al., Cell.Microbial. 7:1418-1431 (2005).

It is now known that azurin, and peptides derived from azurin, such asp28 and p18, have chemopreventive properties. It is now known thatazurin and its derivative, p28, prevent formation of premalignantpreneoplastic lesions in mouse mammary gland organ culture. In a mousemammary gland organ culture model, azurin at 50 μg/ml was found toinhibit the formation of alveolar lesions by 67%. Likewise, p28 at 25μg/ml was found to inhibit the formation of alveolar lesions by 67%. SeeExample 1. Further, azurin at 50 μg/ml was found to inhibit theformation of ductal lesions by 79%, and p28 at 25 μg/ml inhibited theformation of ductal lesions by 71%. See Example 1. Confocal microscopyand FAC showed that azurin and p28 entered normal murine mammaryepithelial cells (MM3MG) and mammary cancer cells (4T1). p28 alsoentered human umbilical vein endothelial cells (HUVEC) in a temperature,time and concentration dependent manner and inhibited capillary tubeformation of HUVEC plated on Matrigel® in a dose dependent manner.Confocal microscopy and FAC also showed that p18 selectively enteredhuman melanoma (Mel-2,7,29), breast (MCF-7), ovarian (SK-OV3),pancreatic (CAPAN-2), glioblastoma (LN-229), astrocytoma (CCF-STTG1),prostate (LN-CAP), and kidney (ACHN-CRL1611) cell lines. In addition,imaging of p18 labeled with an infrared dye (λ_(em) 800 nm) in athymicmice bearing xenografted melanoma tumors clearly demonstrated selectiveuptake in primary s.c. tumors and distant organ metastases withoutaccumulating in normal organs and tissues. It is therefore now knownthat azurin and variants of azurin may be used to inhibit the formationof premalignant preneoplastic lesions, and thus the development ofcancer, and specifically breast cancer, in mammalian patients.

Standard cancer treatment methods, including radiotherapy andchemotherapy, involve damaging the DNA of the cancer cell. The cellularresponse to normal DNA damage includes activation of DNA repair, cellcycle arrest and lethality (Hall, Radiobiology for the Radiologist,Harper and Row, 1988). For example, the induction of DNA double-strandbreaks results in lethal chromosomal aberrations that include deletions,dicentrics, rings, and anaphase bridges (Hall, Radiobiology for theRadiologist, Harper and Row, 1994). Because of the selective uptake ofthe peptides of the present invention by tumors and various cancercells, these peptides, including, in one embodiment, p18, may have useas a non-viral vector for introducing materials into tumors and cancercells. For example, the peptides of the present invention may be used tointroduce DNA or RNA fragments into a cancer cell thereby providing atherapeutic DNA or RNA fragment treatment to a tumor or cancer cell.

Protein transduction domains (PTDs) cluster into two groups based ontheir structural characteristics, cationic residues or amphipathicα-helix, although several fall into both classes. In general, cationicpeptides initially interact with the cell membranes of prokaryotic andeukaryotic species by binding to negatively charged surfaceglycoproteins, facilitating efficient entry into a broad range of normaland malignant cell lines. Kondejewski, L. H., et al., J Biol Chem 277:67-74 (2002); Fuchs, S. M. and Raines, R. T., Biochemistry, 43:2438-2444 (2004). The binding of cationic peptides to HS is consistentwith their high affinity for HS (Kd −109 nM), a value well in excess ofthat reported in the Examples below for azurin, p18 and p28. Tran, D. etal, Proc Natl Acad Sci USA 84: 7957-7961 (1987).

Azurin and peptides derived from it (e.g., p28 and p18) possess theunique property of preferentially entering cancer cells and inhibitingtheir proliferation through cytostatic and cytotoxic mechanisms. Redoxproteins are not normally classified as cell-penetrating peptides(CPPs), or anti-proliferative agents. The entry of azurin, p28, and p18is thought to be distinct from that of cationic CPPs. The amphipathic,azurin fragments p18 and p28 contain the 54-67 amino acid α-helicalstructure of azurin as well as a partial β-sheet structure. AberrantN-glycosylation on several cell surface receptors, including integrinsand cadherins, is associated with changes in progression and metastasisof cancers of diverse histogenesis, suggesting a role for as yet unknownN-glycoslyated cell surface protein(s) in the initial steps of azurin,p18 and p28 penetration. Partridge, E. A., et al, Science 306:120-124(2004); Seales, E. C., et al., Cancer Res 65:4645-4652 (2005).

The temperature dependent entry of cationic CPPs, which supports anendocytotic component to cell penetration, is reflected in the entry ofazurin and amino acid fragment 50-77 of azurin (p28). Yamada, T., etal., Cell Microbiol 7: 1418-1431 (2005). The entry of amino acids 50-67of azurin (p18) into normal and malignant cells appears acceleratedrelative to p28. The lower K_(m) and higher V_(max) of p18 suggest thatamino acids 50-67 of azurin define an amphipathic structure whenassociated with phospholipid membranes that more closely represents theactual PTD of azurin. However, an energy dependent endocytotic or porerelated process does not appear to be the only entry mechanism availableto these peptides. For example, the metabolic and membrane potentialinhibitors sodium azide and ouabain (Na⁺K⁺ ATPase inhibitor), whichinhibit the entry of cationic peptides, did not impair the entry ofeither p18 or p28 into UISO-Mel-2 cells or fibroblasts (FIG. 14 B,C),demonstrating that either peptide may penetrate the cell membranedirectly.

Azurin derived peptides generally use different routes of penetrationcompared to the proposed routes of cellular penetration of othercationic CPPs, i.e., macropinocytosis, distribution to late endosomes orlysosomes along actin filaments or microtubules, and penetration atspecific cell cycle stages, as inhibitors of each of these routes weresingularly ineffective (FIG. 14 B,C). p18, p28 and azurin penetrate theplasma membrane and reach late endosmes, lysosomes and the golgiassociated with caveolae in what has been described as adynamin-independent clathrinin dependent carrier mediated manner.Kirkham, M. and Parton, R. G., Biochem Biophys Acta 1746:349-363 (2005).The striking inhibition of penetration by nocodazole and relative lackof inhibition by cytochalasin-D, which disrupts actin filaments, showscaveolae mediated entry. Id. This route of entry has been described forintegral cell surface components and seemingly disparate molecules,i.e., dextran, and a broad range of pathogens or their products thatalso utilize caveolae to bypass classic endocytic pathways. Depletion ofcholesterol from the plasma membrane with β-methylcylodextran, filipinor nystatin to disrupt lipid rafts, plasma membrane domains that providefluid platforms to segregate membrane components and compartmentalizemembranes, significantly inhibited the penetration of p18 (50%) and p28(˜60%) into UISO-Mel-2 cells and fibroblasts (35% and 42%, respectively)demonstrating that a significant percentage (˜60%) of p18 and p28penetrates the plasma membrane via caveolae. Caveolae are a 50- to100-nm omega-shaped subset of lipid raft invaginations of the plasmamembrane defined by the presence of caveolin specific proteins(caveolin-1, -2, or -3) that function as regulators of signaltransduction.

Brefeldin A disrupts the Golgi apparatus and inhibited p18 accumulation.Thus, this pathway, and the Golgi apparatus, is also utilized in p18 andp28 entry and intracellular transport. Cell penetration of p18 and p28via caveolae comports with the evidence that inhibitors ofN-glycosylation reduce cell entry by 60% in UISO-Mel-2 cells and 25% and35% respectively in fibroblasts. The percentile differences between p18and p28 entry relate to the numbers of N-glycosylation membranestructures in cancer vs normal cells and the relative route of entry ofp28 and p18 via this mechanism. FIG. 14 B, C.

Azurin, p28, and p18 all bind to cancer cells with high affinity andhigh capacity relative to many other potential anti-cancer peptides.After binding, this protein/receptor complex localizes in caveolae andis internalized, eventually moving (via caveosomes) to the golgi, ER,and nucleus. In addition to caveolar-mediated entry, kinetic analysisalso demonstrates that p28 and p18 penetrate the plasma membrane via anon-clathrin caveolae mediated process. A clathrin- andcaveolin-independent pathway exists as a constitutive internalizationmechanism, such as for the interleukin 2 receptor and for certainglycosyl-phosphatidylinositol (GPI)-anchored proteins. Lamaze, C., etal., Mol Cell 7: 661-671 (2001); Sabharanjak, S., et al., Dev Cell, 2:411-423 (2002). Clathrin- and caveolin-independent endocytosis is alsoused by pathogens to invade cells, either exclusively, as for the murinepolyoma virus, or in combination with a conventional pathway, as is thecase for the influenza virus. Ewers, H., et al, Proc Natl Acad Sci USA102: 15110-15115 (2005); Sieczkarski, S. B. and Whittaker, G. R., JVirol, 76: 10455-10464 (2002). An increase in caveolin-1 expression incancer cells over normal cells is not likely to be the sole basis forthe preferential entry of azurin, p28 and p18 into cancer cells.Fibroblasts and a number of other normal cells also have significantnumbers of caveolae on their surface.

Examples 18-24 show that p18 (amino acids 50-67 of azurin) and p28(amino acids 50-77 of azurin) preferentially penetrate cancer cells viaendocytotic, caveosome directed and caveosome independent pathways. Thecellular penetration of p18 and p28 is unique relative to all currentCPPs in its preference for cancer cells. Surprisingly, the C-terminal10-12 amino acids of p28 (SEQ ID NOS: 35, 36, and 37) comprise a domainprimarily responsible for cell cycle inhibition and apoptoticactivity/cytotoxicity. Furthermore, this same domain is most likely tocontact specific residues on a cell membrane regardless of the cell'sstatus and thus facilitate entry; amino acids 69, 70, 75, 76, and 85 ofazurin in particular provide contact to the cell membrane. Peptides withthe same amino acids or amino acids with similar structure located atthe same positions in the peptide chain, or positions in the peptidechain that are similar or equivalent to those of amino acids 69, 70, 75,76, and 85 of azurin, should have the same or similar ability to contactspecific cell membrane residues and enter cells. Once internalized, p28inhibits cancer cell proliferation initially through a cytostaticmechanism. Thus, p18 and p28 account for the preferential entry ofazurin into human cancer cells and a significant amount of theanti-proliferative and cytotoxic activity of azurin on human cancercells, respectively.

In addition to entering cancer cells, p18 and p28 are able to entertumors and mammalian organs, as is shown in FIGS. 16 through 53, whichwere obtained using the methods disclosed in Example 31. Surprisingly,p18 and p28 are also able to penetrate the blood-brain barrier and entermammalian brains, as demonstrated by, for example, FIGS. 20A, 20B, 21B,23, 24, 28, 30, 31A, 32B, 33, 34B, 35A-B, 37A-C, 38A-C, 40A, 42A, 43A,45A-D, 46, 47B, 48A-B, and 50.

The peptides of the present invention can be used to introduce othermolecules or compounds, such as DNA or RNA fragments, into mammaliancancer cells. The following describe non-limiting exemplary techniquesand/or particular DNA or RNA fragments that can be introduced with thepeptides of the present invention, and, in one embodiment, p18, whichfacilitate the entry of a linked molecule into a mammalian cancer cell.For example, the compounds of the invention, which may preferentiallyenter cells, can be used with gene therapy, RNAi approaches,hematopoietic gene transfer, homologous recombination, ribozymetechnology, antisense technology, tumor immunotherapy and tumorsuppressors, translational research, anti-gene therapy (antisense, siRNA& ribozymes), apoptosis, immunology and immunotherapy, DNA synthesis andrepair.

Gene therapy involves the transfer of a foreign gene into a cancer cell,for example a tumor suppressor or inducer of apoptosis, under conditionssuitable for expression of the gene. Once expressed, the gene productconfers a beneficial effect on the tumor cell by either slowing itsgrowth, inhibiting its metastatic potential, or killing it outright.Historically, the clinical effectiveness of cancer gene therapy has beenlimited by 1) lack of control of therapeutic gene expression within thetumor, and 2) selective targeting of the vector to the tumor. Thecompounds of the present invention address the selective targeting oftumor cells. Moreover, several strategies have been proposed for thecontrol of gene expression. One strategy is transcriptional targeting inwhich the promoter regulating the therapeutic gene is activated bytumor-selective transcription factors. Examples include the use of theMUC-1 promoter in breast cancer and the CEA promoter in colon cancer(Kurihara et al., “Selectivity of a replication-component adenovirus forhuman breast carcinoma cells expressing the MUC1 antigen,” J. Clin.Invest. 106(6): 763-771, 2000; Konishi et al., “Transcriptionallytargeted in vivo gene therapy for carcinoembrionic antigen-producingadenocarcinoma,” J. Med. Sci., 48(3): 79-89, 1999).

Antisense techniques rely on the introduction of a nucleic acid moleculeinto a cell which typically is complementary to a mRNA expressed by theselected gene. The antisense molecule typically suppresses translationof the mRNA molecule and prevents the expression of the polypeptideencoded by the gene. Modifications of the antisense technique mayprevent the transcription of the selected gene by the antisense moleculebinding to the gene's DNA to form a triple helix; One particularantisense drug that can be used in accordance with the present inventionis G3139 (also known as oblimersen; manufactured by Genta, Inc.,Lexington, Mass.). Another particular antisense molecule that can beused is G4460 (also known as c-myb antisense manufactured by Genta,Berkeley Heights, N.J.).

RNA interference (RNAi) based molecules can also be attached to thepeptides of the present invention. RNAi is generally mediated by doublestranded RNA (“dsRNA”), short hairpin RNA (“shRNA”) or other nucleicacid molecules with similar characteristics. These nucleic acidmolecules are processed or cut into smaller pieces by cellular enzymesincluding Dicer and Drosha. The smaller fragments of the nucleic acidmolecules can then be taken up by a protein complex (the RISC complex)that mediates degradation of mRNAs. The RISC complex will degrade mRNAthat complementarily base pairs with the nucleic acid molecules it hastaken up. In this manner, the mRNA is specifically destroyed, thuspreventing encoded-for proteins from being made.

Ribozyme technologies rely on the introduction of a nucleic acidmolecule into a cell which expresses a RNA molecule which binds to, andcatalyses the selective cleavage of, a target RNA molecule. The targetRNA molecule is typically a mRNA molecule, but it may be, for example, aretroviral RNA molecule.

Targeted gene deletion by homologous recombination, which requires twogene-inactivating events (one for each allele) is also a strategy thatcan be used with the present invention.

Particular therapies delivered in conjunction with the compounds of thepresent invention can also be directed against cancer-specifictranscription complexes (CSTCs) that can control expression of proteinsthat are critical for cancer development. See, for example, UnitedStates Patent Application No. 2008/0027002 which is incorporated byreference herein for its teachings regarding cancer therapies directedagainst CSTCs.

Due to the high degree of structural similarity between cupredoxins, itis likely that cupredoxins other than azurin and truncations thereofwill also be able to preferentially enter cancer cells vianon-endocytotic and endocytotic pathways, and will further be able toinhibit the formation of premalignant lesions in mammals. Suchcupredoxins may be found in, for example, bacteria or plants. Severalcupredoxins are known to have pharmacokinetic activities similar tothose of azurin from Pseudomonas aeruginosa. For example, rusticyaninfrom Thiobacillus ferrooxidans (SEQ ID NO: 4) can also enter macrophagesand induce apoptosis. Yamada et al., Cell Cycle 3:1182-1187 (2004);Yamada et al., Cell. Micro. 7:1418-1431 (2005). Plastocyanin fromPhormidium laminosum (SEQ ID NO: 3) and pseudoazurin form Achromobactercycloclastes (SEQ ID NO: 5) also are cytotoxic towards macrophages. U.S.Pat. Pub. No. 20060040269, published Feb. 23, 2006. It is thereforecontemplated that other cupredoxins may be used in the compositions andmethods of the invention. Further, variants, derivatives, truncations,and structural equivalents of cupredoxins that retain the ability toinhibit the formation of cancer in mammals may also be used in thecompositions and methods of the invention. These variants andderivatives may include, but are not limited to, truncations of acupredoxin, conservative substitutions of amino acids and proteinmodifications disclosed herein, including but not limited to PEGylationand all-hydrocarbon stabling of α-helices.

Chemoprevention Through p53

The interaction of amino acids 50-77 of azurin (p28, SEQ ID NO: 2) andp53 was studied and is described in Examples 26 to 30 below. Asdisclosed herein, p28 penetrates and exhibits an anti-proliferativeeffect on human breast cancer cells that is mediated by p53, a tumorsuppressor protein that becomes functionally active in response tostress and triggers either cell cycle arrest or cell death. Experimentsusing a series of GST-pull down assays, glycerol gradientcentrifugation, microcalorimetric experiments, single molecule forcespectroscopy, and computer modeling show that azurin binds within eitherthe N-terminal or DNA binding domains of p53 and increases itsintracellular levels. The results disclosed in Examples 26 to 30 andFIGS. 54-57 herein refine the binding site(s) for p28 to within aminoacids 1-17, 24-31, 80-276 or 297-305, the N-terminal and DNA bindingdomains of p53.

Suggestions that the azurin binding domain for p53 includes ahydrophobic patch described by azurin Met44 and Met64 are supported byevidence that a disrupted hydrophobic patch mutant (mutant azurinM44KM64E) is less cytotoxic to human melanoma (Mel-2) cells than wtazurin. This shows that the p53 binding domain of the azurin moleculesurrounds the hydrophobic patch. A recent docking simulation studydemonstrated a significant loss of ˜75 kJ/mol in the interaction freeenergy of the mutant complex with respect to wild type azurin, againindicating that the hydrophobic patch of azurin surrounding residuesMet44 and Met64 is important for interaction with p53. As Met64 resideswithin the p53 binding site of p28 (amino acid 15 of p28), competitionassays, mutant studies, and docking experiments clearly show that thisis the azurin domain that binds to p53.

The tumor suppressor protein p53 is a predominantly nuclear protein thatacts as a transcriptional regulator for many genes, including the 21 kDaprotein p21/Waf1/Cip1, an inhibitor of cell cycle progression. Treatmentof MCF-7 cells with p28 increased p53 levels, leading to higherintracellular levels of p21, a strong inhibitor of cyclin dependentkinase (CDK) activity, especially cdc2 and CDK2 that regulate cell cycleprogression at G₁ and G₂/M, respectively. In the progression through theG₂/M phase, cdc2 and CDK2 kinases are activated primarily in associationwith cyclin B and cyclin A, respectively. The CDK inhibitor p21associates efficiently with cyclin A in G₂/M arrested cells, althoughunder the same conditions, cyclin B1 does not associate with p21 and thelevel of cyclin B1 increases continuously. This shows that the p28induced G₂/M arrest in MCF-7 cells is associated with inhibition of CDK2and cyclin A (FIG. 57A).

The p28-induced increase in p21 in MCF-7 cells was also accompanied by atime-dependent increase in p27, another member of the Cip/Kip CDKIfamily. Hsu et al., 2008 recently demonstrated that induction of p53increased both p21 and p27 promoter activity as determined by luciferaseassay. Cellular and Molecular Life Sciences (CMLS), 2008. In addition,p53 DNA binding activity of the p21 and p27 promoters is activated bythe p53 inducer, progesterone, which means that not only p21, but alsop27 is transcriptionally regulated by p53. Collectively, data shows thatp28 enhancement of p53 levels subsequently up-regulates p21 and p27,inducing a significant decrease in intracellular CDK2 and cyclin Alevels in MCF-7 cells and inhibition of the cell cycle at G₂/M (FIG.57A). The reported lack of or inefficient association between cyclin B1and p21, suggests the increase in cyclin B/cdc2 activity followingexposure to p28 may reflect a similar pattern following a p28 inducedincrease in p21. An increase in phosphorylated cdc2 (inactive form)following exposure to p28 accompanied the increased cellular level ofcyclin B1, suggesting the increase in the cdc2-cyclin B complex isreflected by the increase in cdc2 phosphorylation. A similar G₂ arrestin MCF-7 and MDA-MB-468 human breast cancer cells, accompanied by highlevels of cytoplasmic cyclin B1, is induced by nocodazole, a knowndisruptor of microtubules, and transcriptional and translationalactivator of p21. Differentiation agents such as all-trans retinoic acid(ATRA) and sodium butyrate (SB) produce a similar phenomenon of growthinhibition and G₁ arrest in oral squamous carcinoma cells thatcorrelates with the induction of G₁ phase cell cycle regulatory proteinsCDK6, p21 and p27, and the inhibition of the G₂ phase cell cycleregulatory protein CDK2. Since p28 did not enhance p21 in MDD2 cells,and p27 appears absent in these cells, the levels of CDK2 and cyclin Awere not significantly altered (FIG. 57B) and no inhibition of cellcycle occurred. Additional evidence for a p28 induced decrease in theCDK2 and cyclin A complex, a key regulator of cdc2 activity in humancells, causing a G₁ and G₂/M arrest is found in the G₂ delay thatfollows cyclin A RNAi introduction to HeLa cells, which inactivates theCDK2-cyclin A complex causing cell cycle arrest in G₂/M.

Although Cip/Kip family proteins such as p21 and p27 are potentinhibitors of cyclin A dependent CDK2, they also act as positiveregulators of cyclin D-dependent kinases. Cip/Kip family proteins canstabilize CDK4 and CDK6. CDK4 is amplified and overexpressed in widevariety of tumors including breast, gliomas, sarcomas and carcinomas ofthe uterine cervix, whereas the CDK6 gene is amplified in certain typeof malignancies including squamous cell carcinomas, gliomas and lymphoidtumors. Although, the initial or control level of CDK6 is lower thanCDK4, CDK6, but not CDK4 levels are continuously elevated in MCF-7 cellsexposed to p28. Again, there was no alteration in CDK4 and CDK6 in MDD2cells where p53 and p21 did not increase in response to p28 (FIG. 57B).The Ink4 group, p16^(Ink4a), p15^(Ink4b), p18^(Ink4c) and p19^(Ink4d) ofCDKIs specifically associates with and inhibits CDK4 and CDK6 whichregulate cell cycle progression at G₁. Since the p16^(Ink4a) gene ishomozygously deleted in MCF-7 cells, Ink4 CDKI proteins should exhibitless of an inhibitory effect on CDK6 than CDK4, providing a rationalefor the increase on CDK6 observed in CDK6 in the presence of essentiallystable CDK4 levels.

Collectively, these results demonstrate that p28 binds to p53,increasing p53 levels that subsequently amplify anti-proliferativeactivity through p21 and p27 inactivation of the CDK2-cyclin A complex,causing a G₂/M cell cycle arrest in MCF-7 breast cancer cells in vitroand inhibition MCF-7 xenograft growth in athymic mice.

Compositions of the Invention

In certain embodiments, the invention provides for peptides that arecupredoxin(s) or variants, truncations, derivatives or structuralequivalents of cupredoxin that inhibit the development of premalignantlesions in mammalian cells, tissues and animals. In other aspects, theinvention further provides for peptides that are cupredoxin(s) orvariants, truncations, derivatives or structural equivalents ofcupredoxin that inhibit the development of cancer in mammalian cells,tissues and animals. In some embodiments, the peptide comprises theC-terminus of p28 (SEQ ID NO: 2), such as SEQ ID NO. 35, SEQ ID NO. 36,or SEQ ID NO. 37. In other embodiments, the peptide comprises one ormore of the amino acids located at positions 69, 70, 75, 76, and 85 ofSEQ ID NO: 1 in locations the same or similar to those of azurin.

Some embodiments of the invention further provide for peptides that arecupredoxin(s) or variants, truncations, derivatives or structuralequivalents of cupredoxin that preferentially enter cancer cells. Infurther aspects, the invention provides for peptides that arecupredoxin(s) or variants, truncations, derivatives or structuralequivalents of cupredoxin that preferentially enter cells by endocytoticpathways, including caveolae-mediated and Golgi mediated pathways, andhave chemopreventive effects therein. In further embodiments, thepeptide comprises or consists of the C-terminus of p28, such as SEQ IDNO. 35, SEQ ID NO. 36, or SEQ ID NO. 37. In other embodiments, thepeptide comprises one or more of the amino acids located at positions69, 70, 75, 76, and 85 of SEQ ID NO: 1 in locations the same or similarto those of azurin. In other embodiments, the peptide comprises theamino acids located at 69, 70, 75, 76, and 85 of SEQ ID NO. 1 inlocations the same or similar to those of azurin.

In some embodiments, the peptide is isolated. In some embodiments, thepeptide is substantially pure or pharmaceutical grade. In otherembodiments, the peptide is in a composition that comprises, or consistsessentially of, the peptide. In another specific embodiment, the peptideis non-antigenic and does not raise an immune response in a mammal, andmore specifically a human. In some embodiments, the peptide is less thata full-length cupredoxin, and retains some of the pharmacologicactivities of the cupredoxins. Specifically, in some embodiments, thepeptide may retain the ability to inhibit the development ofpremalignant lesions in the mouse mammary gland organ culture. In otherembodiments, the peptide retains the ability to directly andpreferentially enter cells via, for example, caveolae-mediatedendocytosis. In other embodiments, the peptide retains the ability todirectly and preferentially enter cells and have chemopreventive effectstherein.

In other aspects, the invention also provides compositions comprising atleast one peptide that is a cupredoxin, or variant, derivative,truncation or structural equivalent of a cupredoxin that canpreferentially enter cancer cells, specifically in a pharmaceuticalcomposition. In specific embodiments, the pharmaceutical composition isdesigned for a particular mode of administration, for example, but notlimited to, oral, intraperitoneal, or intravenous. Such compositions maybe hydrated in water, or may be dried (such as by lyophilization) forlater hydration. Such compositions may be in solvents other than water,such as but not limited to, alcohol.

Certain embodiments of the invention also provide compositionscomprising peptides that are variants, derivatives, truncations orstructural equivalents of cupredoxin that preferentially enter cancercells and/or tumors in mammalian cells, tissues and animals. In someembodiments, the peptide is the C-terminus of p28, such as SEQ ID NO. 35or SEQ ID NO. 36. In some embodiments, the peptide is p18 having SEQ IDNO. 25. In some embodiments, the peptide is a variant, derivative orstructural equivalent of p18. In some embodiments, the composition isp18 coupled to DNA or RNA. In some embodiments, the DNA or RNA is a geneor a portion of a gene. In some embodiments, the DNA or RNA has atherapeutic effect once delivered. In some embodiments, the peptide isp28 having SEQ ID NO. 2. In some embodiments, the peptide is a variant,derivative, or structural equivalent of p28. In some embodiments, thecomposition is p28 coupled to DNA or RNA. In some embodiments, the DNAor RNA is a gene or a portion of a gene. In some embodiments, the DNA orRNA has a therapeutic effect once delivered.

Because of the high structural homology between the cupredoxins, it iscontemplated that cupredoxins will have the same chemopreventiveproperties as azurin and p28. In some embodiments, the cupredoxin is,but is not limited to, azurin, pseudoazurin, plastocyanin, rusticyanin,auracyanin, stellacyanin, cucumber basic protein or Laz. In particularlyspecific embodiments, the azurin is derived from Pseudomonas aeruginosa,Alcaligenes faecalis, Achromobacter xylosoxidans ssp. denitrificans I,Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitides,Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis,Xylella fastidiosa, Ulva pertussis or Vibrio parahaemolyticus. In oneembodiment, the azurin is from Pseudomonas aeruginosa. In other specificembodiments, the cupredoxin comprises an amino acid sequence that is SEQID NO: 1, 3-19.

Aspects of the invention include peptides that are amino acid sequencevariants which have amino acids replaced, deleted, or inserted ascompared to the wild-type cupredoxin. Variants of the invention may betruncations of the wild-type cupredoxin. In some embodiments, thepeptide of the invention comprises a region of a cupredoxin that is lessthat the full length wild-type polypeptide. In some embodiments, thepeptide of the invention comprises more than about 10 residues, morethan about 15 residues or more than about 20 residues of a truncatedcupredoxin. In some embodiments, the peptide comprises not more thanabout 100 residues, not more than about 50 residues, not more than about40 residues, not more than about 30 residues or not more than about 20residues of a truncated cupredoxin. In some embodiments, a cupredoxinhas to the peptide, and more specifically SEQ ID NOS: 1, 3-19 as to thepeptide of the invention, at least about 70% amino acid sequenceidentity, at least about 80% amino acid sequence identity, at leastabout 90% amino acid sequence identity, at least about 95% amino acidsequence identity or at least about 99% amino acid sequence identity.

In specific embodiments, the variant of cupredoxin comprises P.aeruginosa azurin residues 50-77 (p28, SEQ ID NO: 2), azurin residues50-67 (p18, SEQ ID NO: 25), or azurin residues 36-88 (SEQ ID NO: 26). Inother embodiments, the variant of cupredoxin consists of P. aeruginosaazurin residues 50-77 (SEQ ID NO: 2), azurin residues 50-67 (SEQ ID NO:25), or azurin residues 36-88 (SEQ ID NO: 26). In other specificembodiments, the variant consists of the equivalent residues of acupredoxin other that azurin. It is also contemplated that othercupredoxin variants can be designed that have a similar pharmacologicactivity to azurin residues 50-77 (SEQ ID NO: 2), or azurin residues36-88 (SEQ ID NO: 26). To do this, the subject cupredoxin amino acidsequence will be aligned to the Pseudomonas aeruginosa azurin sequenceusing BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residueslocated on the P. aeruginosa azurin amino acid sequence, and theequivalent residues found on the subject cupredoxin sequence, and theequivalent peptide thus designed.

In one embodiment of the invention, the cupredoxin variant contains atleast amino acids 57 to 89 of auracyanin B of Chloroflexus aurantiacus(SEQ ID NO: 20). In another embodiment, the cupredoxin variant containsat least amino acids 50-67 of Pseudomonas aeruginosa azurin (SEQ ID NO25). In another embodiment of the invention, the cupredoxin variantcontains at least amino acids 51-77 of Pseudomonas syringae azurin (SEQID NO: 21). In another embodiment of the invention, the cupredoxinvariant contains at least amino acids 89-115 of Neisseria meningitidisLaz (SEQ ID NO: 22). In another embodiment of the invention, thecupredoxin variant contains at least amino acids 52-78 of Vibrioparahaemolyticus azurin (SEQ ID NO: 23). In another embodiment of theinvention, the cupredoxin variant contains at least amino acids 51-77 ofBordetella bronchiseptica azurin (SEQ ID NO: 24).

The variants may also include peptides made with synthetic amino acidsnot naturally occurring. For example, non-naturally occurring aminoacids may be integrated into the variant peptide to extend or optimizethe half-life of the composition in the bloodstream. Such variantsinclude, but are not limited to, D,L-peptides (diastereomer), (forexample Futaki et al., J. Biol. Chem. 276(8):5836-40 (2001); Papo etal., Cancer Res. 64(16):5779-86 (2004); Miller et al, Biochem.Pharmacol. 36(1):169-76, (1987).; peptides containing unusual aminoacids (for example Lee et al., J. Pept. Res. 63(2):69-84 (2004)),olefin-containing non-natural amino acid followed by hydrocarbonstapling (for example Schafmeister et al., J. Am. Chem. Soc.122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)),and peptides comprising ε-(3,5-dinitrobenzoyl)-Lys residues.

In other embodiments, the peptide of the invention is a derivative of acupredoxin. The derivatives of cupredoxin are chemical modifications ofthe peptide such that the peptide still retains some of its fundamentalactivities. For example, a “derivative” of azurin can be a chemicallymodified azurin that retains its ability to preferentially enter cellsvia endocytotic or non-endocytotic pathways, as well as the ability toinhibit the development of premalignant lesions in mammalian cells,tissues or animals. Chemical modifications of interest include, but arenot limited to, hydrocarbon stabling, amidation, acetylation, sulfation,polyethylene glycol (PEG) modification, phosphorylation andglycosylation of the peptide, and other methods disclosed herein. Inaddition, a derivative peptide maybe a fusion of a cupredoxin, orvariant, derivative or structural equivalent thereof to a chemicalcompound, such as but not limited to, another peptide, drug molecule orother therapeutic or pharmaceutical agent or a detectable probe.Derivatives of interest include chemical modifications by which thehalf-life in the bloodstream of the peptides and compositions of theinvention can be extended or optimized, such as by several methods wellknown to those in the art, including but not limited to, circularizedpeptides (for example Monk et al., BioDrugs 19(4):261-78, (2005);DeFreest et al., J. Pept. Res. 63(5):409-19 (2004)), N- and C-terminalmodifications (for example Labrie et al., Clin. Invest. Med.13(5):275-8, (1990)), and olefin-containing non-natural amino acidfollowed by hydrocarbon stapling (for example Schafmeister et al., J.Am. Chem. Soc. 122:5891-5892 (2000); Walenski et al., Science305:1466-1470 (2004)).

In some embodiments, the cupredoxin may be changed using methods thatinclude, but are not limited to, those which decrease the hydrolysis ofthe peptide, decrease the deamidation of the peptide, decrease theoxidation, decrease the immunogenicity and/or increase the structuralstability of the peptide. In some embodiments, the cupredoxin may bemodified using methods that enhance its ability to preferentially entercancer cells and/or have cytotoxic effects therein. It is contemplatedthat two or more of the modifications described herein may be combinedin one modified cupredoxin derived peptide, as well as combinations ofone or more modifications described herein with other modification toimprove pharmacokinetic properties that are well know to those in theart. Many methods to design such variants and derivatives are well knownin the art.

Biotransformation

One approach to improving the pharmacokinetic properties of peptides isto create variants and derivatives of the cupredoxin derived peptidesthat are less susceptible to biotransformation. Biotransformation maydecrease the pharmacologic activity of the peptide as well as increasethe rate at which it is eliminated from the patient's body. One way ofachieving this is to determine the amino acids and/or amino acidsequences that are most likely to be biotransformed and to replace theseamino acids with ones that are not susceptible to that particulartransformative process.

In some embodiments, the cupredoxin derived peptides may includeunnatural amino acids or modified amino acids. In some embodiments, theintroduction of certain unnatural amino acids enhances thepharmcaokinetic properties of the cupredoxin derived peptide. Suchintroduction may be site-specific and may be done to avoid certainbiochemical modifications in vivo. Exemplary unnatural amino acidsinclude b-amino acids (e.g., b3 and b2), homo-amino acids, cyclic aminoacids, aromatic amino acids, Pro and Pyr derivatives, 3-substitutedAlanine derivatives, Glycine derivatives, Ring-substituted Phe and TyrDerivatives, Linear Core Amino Acids and Diamino Acids. Such unnaturalamino acids may be incorporated into peptides by site directedmodification, ribosomal translation, or by chemical synthesis of thepeptide. Each of these methods may be applied in synthesizing cupredoxinderived peptides.

For example, modified cupredoxin derived peptides may be synthesized bythe use of wild-type Aminoacyl-tRNA synthetases (AARSs) with unnaturalamino acids building for the production of unnatural cupredoxinvariants. See Hartman, et al., PLoS One, 2(10): e972 (2007); Miranda, etal., J. Am. Chem. Soc. 129: 13153-13159 (2007). The specificity of theribosomal translation apparatus limits the diversity of unnatural aminoacids that may be incorporated into peptides using ribosomaltranslation. Over ninety unnatural building blocks that are AARSsubstrates have been uncovered including side chain and backboneanalogs. Hartman, et al., PLoS One, 2(10): e972 (2007). Over fiftyunnatural amino acids may be incorporated into peptides with highefficiency using an all-enzymatic translation system, with peptidescontaining up to thirteen different unnatural amino acids. Hartman, etal., PLoS One, 2(10): e972 (2007). In some embodiments, such amino acidsmay be incorporated in cupredoxin derived peptides.

One method of chemically modifying a cupredoxin or cytochrome c551 orvariant, derivative, truncation, or structural equivalent thereof may beto follow the steps taken to design an anti-HIV small protein, CCL-5(RANTES) with improved pharmaceutical properties by, for example,hydrophobic N-terminal modification, total protein-polymer conjugatechemicals synthesis, coded and noncoded amino acid mutagenesis, peptidebackbone engineering, and site-specific polymer attachment. Anti-HIVproteins can be designed by incorporating natural and unnatural aminoacid residues into CCL-5 analogues baring polymer substituents atvarying attachment positions. Studies indicate that in vitro anti-HIVactivity of polymer-modified CCL-5 derivatives correlates with CCR-5signaling, so changes to the peptide should not disrupt CCR-5 activity.Miranda, et al., J. Am. Chem. Soc. 129: 13153-13159 (2007), thedisclosure of which is incorporated in its entirety herein.

Other modifications may include the use of optically active α-aminoacids. The use of optically active α-amino acids and their derivativesis being expanded for their use in pharmaceuticals, agrochemicals and aschiral ligands. In particular, chiral glycine and alanine equivalentsplan an important role. At least one stereoselective strategy forconstructing α-amino acids has been proposed, allowing for enantiopureα-amino acids in predetermined stereochemistry. Lu, et al. “AsymmetricSynthesis of α-amino acids: Preparation and alkylation of monocycliciminolactones derived from α-Methyl trans-cinnamaldehyde” published onInternet on Sep. 11, 2008 (to be published in J. Org. Chem.), thedisclosure of which is incorporated by reference herein. The modifiedcupredoxin derived peptides may be synthesized using the opticallyactive α-amino acids to produce enantiomerically enriched iterations.

Hydrolysis is generally a problem in peptides containing aspartate.Aspartate is susceptible to dehydration to form a cyclic imideintermediate, causing the aspartate to be converted to the potentiallyinactive iso-aspartate analog, and ultimately cleaving the peptidechain. For example, in the presence of aspartic acid—proline in thepeptide sequence, the acid catalyzed formation of cyclic imideintermediate can result to cleavage of the peptide chain. Similarly, inthe presence of aspartic acid—glycine in the peptide sequence, thecyclic intermediate can be hydrolyzed either into the original aspartateform (harmless) or into the iso-aspartate analog. Eventually, all of theaspartate form can be completely converted into the iso-aspartateanalog. Similarly sequences with serine can also be dehydrated to form acyclic imide intermediate that can cleave the peptide chain. Cleavage ofthe peptide may result in reduced plasma half-life as well as reducedspecific pharmacologic activity of the peptide.

It is contemplated that substituting other amino acids for asparagineand/or serine in the sequence of the cupredoxin derived peptide mayresult in a peptide with improved pharmacokinetic properties such as alonger plasma half-life and increased specific activity of apharmacologic activity of the peptide. In one contemplated variant, atone or more asparagine residues of the cupredoxin derived peptide may bereplaced with another amino acid residue, and specifically a glutamicacid residue. In another contemplated variant, one or more serineresidues of the cupredoxin derived peptide may be replaced with anotheramino acid residue, and specifically a threonine residue. In somevariants of cupredoxin derived peptide, one or more asparagine residuesand one or more serine residues are substituted. In some embodiments,conservative substitutions are made. In other embodiments,non-conservative substitutions are made.

Deamidation of amino acid residues is a particular problem inbiotransformation. This base-catalyzed reaction frequently occurs insequences containing asparagine-glycine or glutamine-glycine and followsa mechanism analogous to the aspartic acid-glycine sequence above. Thede-amidation of the asparagine-glycine sequence forms a cyclic imideintermediate that is subsequently hydrolyzed to form the aspartate oriso-asparate analog of asparagine. In addition, the cyclic imideintermediate can lead to racemization into D-aspartic acid orD-iso-aspartic acid analogs of asparagine, all of which can potentiallylead to inactive forms of the peptide.

It is contemplated that deamidation in the cupredoxin peptides may beprevented by replacing a glycine, asparagine and/or glutamine of theasparagine—glycine or glutamine—glycine sequences of the cupredoxin withanother amino acid and may result in a peptide with improvedpharmacokinetic properties, such as a longer plasma half-life andincreased specific activity of a pharmacologic activity of the peptide.In some embodiments, the one or more glycine residues of the cupredoxinderived peptide are replaced by another amino acid residue. In specificembodiments, one or more glycine residues of the cupredoxin derivedpeptide are replaced with a threonine or an alanine residue. In someembodiments, the one or more asparagine or glutamine residues of thecupredoxin derived peptide are replaced by another amino acid residue.In specific embodiments, one or more asparagine or glutamine residues ofthe cupredoxin derived peptide are replaced with an alanine residue. Inother specific embodiments, the glycine at residues 58 and/or 63 of P.aeruginosa azurin (SEQ ID NO: 1), or equivalent glycines of othercupredoxins, are replaced with an alanine or a threonine. In otherspecific embodiments, the methionine at residue 59 of P. aeruginosaazurin (SEQ ID NO: 1), or an equivalent methionine residue of anothercupredoxin derived peptide, is replaced by an alanine residue. In otherspecific embodiments, the glycine at residue 63 of P. aeruginosa azurin(SEQ ID NO: 1), or an equivalent glycine residue of another cupredoxinderived peptide, is replaced by an threonine residue. In someembodiments, conservative substitutions are made. In other embodiments,non-conservative substitutions are made. In specific embodiments, themodified cupredoxin derived peptide of the invention comprises thefollowing sequence, wherein the underlined amino acids are substitutedinto the wildtype Pseudomonas aeruginosa p28 sequence

LSTAADMQAVVTDTMASGLDKDYLKPDD (SEQ ID NO: 38)

Reversible and irreversible oxidation of amino acids are otherbiotransformative processes that may also pose a problem that may reducethe pharmacologic activity, and/or plasma half-life of cupredoxinderived peptides. The cysteine and methionine residues are thepredominant residues that undergo reversible oxidation. Oxidation ofcysteine is accelerated at higher pH, where the thiol is more easilydeprotonated and readily forms intra-chain or inter-chain disulfidebonds. These disulfide bonds can be readily reversed in vitro bytreatment with dithiothreitol (DTT) or tris(2-carboxyethylphosphine)hydrochloride (TCEP). Methionine oxidizes by both chemical andphotochemical pathways to form methionine sulfoxide and further intomethionine sulfone, both of which are almost impossible to reverse.

It is contemplated that oxidation in the cupredoxin derived peptides maybe prevented by replacing methionine and/or cysteine residues with otherresidues. In some embodiments, one or more methionine and/or cysteineresidues of the cupredoxin derived peptide are replaced by another aminoacid residue. In specific embodiments, the methionine residue isreplaced with a leucine or valine residue. In other specificembodiments, one or more of the methionines at residues 56 and 64 of P.aeruginosa azurin (SEQ ID NO: 1), or equivalent methionine residues inother cupredoxin derived peptides, are replaced with leucine or valine.In some embodiments, conservative substitutions are made. In otherembodiments, non-conservative substitutions are made. In specificembodiments, the cupredoxin peptides of the invention comprise one ofthe following sequences, wherein the underlined amino acid issubstituted into the wildtype Pseudomonas aeruginosa p28 sequence:

LSTAADLQGVVTDGLASGLDKDYLKPDD (SEQ ID NO: 39) orLSTAADVQQVVTDGVASGLDKDYLKPDD. (SEQ ID NO: 40)

Another biotransformative process that may affect the pharmacologicactivity, (such as the ability to preferentially enter cells), plasmahalf-life and/or immunogenicity of the cupredoxin derived peptides isdiketopiperazine and pyroglutamic acid formation. Diketopiperazineformation usually occurs when glycine is in the third position from theN-terminus, and more especially if proline or glycine is in position 1or 2. The reaction involves nucleophilic attack of the N-terminalnitrogen on the amide carbonyl between the second and third amino acid,which leads to the cleavage of the first two amino acids in the form ofa diketopiperazine. On the other hand, pyroglutamic acid formation maybe almost inevitable if glutamine is in the N-terminus. This is ananalogous reaction where the N-terminal nitrogen attacks the side chaincarbonyl carbon of glutamine to form a deaminated pyroglutamayl peptideanalog. This conversion also occurs in peptide containing asparagine inthe N-terminus, but to a much lesser extent.

It is contemplated that diketopiperazine and pyroglutamic acid formationmay be decreased in cupredoxin derived peptides by replacing glycine inposition 1, 2, or 3 from the N-terminus, proline in position 3 from theN-terminus, or asparagine at the N-terminus of the peptide with anotheramino acid residue. In some embodiments, a glycine in positions 1, 2, or3 from the N-terminus of the cupredoxin derived peptide is replaced withanother amino acid residue. In specific embodiments, the glycine residueis replaced by a threonine or alanine residue. In another embodiment, aproline at position 3 from the N-terminus of the cupredoxin derivedpeptide is replaced with another amino acid residue. In specificembodiments, the proline is replaced by an alanine residue. In anotherembodiment, an asparagine at the N-terminus is replaced with anotheramino acid residue. In specific embodiments, the asparagine residue isreplaced by a glutamine residue. In some embodiments, conservativesubstitutions are made. In other embodiments, non-conservativesubstitutions are made.

Another biotransformative process that may affect the pharmacologicactivity, plasma half-life and/or immunigenicity of the cupredoxinderived peptide is racemization. This term is loosely used to refer tothe overall loss of chiral integrity of the amino acid or peptide.Racemization involves the base-catalyzed conversion of one enantiomer(usually the L-form) of an amino acid into a 1:1 mixture of L- andD-enantiomers. One way to improve stability of the peptide in general isby making a retro-inverso (D-isomer) peptide. The double inversion ofpeptide structure often leaves the surface topology of the side-chainintact and has been used extensively to stabilize biologically activepeptides. Snyder et al., PLoS Biol. 2:0186-0193 (2004). A D-amino acidsubstituted Tat is internalized into cells as well as the L-amino acidpeptide. Futaki et al., J. Biol. Chem. 276:5836-5840 (2001); Huq et al.,Biochemistry 38:5172-5177 (1999). In some embodiments, one or more aminoacid residues of the cupredoxin derived peptide are replaced by theD-isomer of that amino acid residue. In other embodiments, all of theamino acid residues of the cupredoxin derived peptide are replaced withD-isomers of those residues. In one embodiment, the modified cupredoxinderived peptide is a retro-inverso (D-isomer) version of the cupredoxinderived peptide. In a specific embodiment, the modified cupredoxinderived peptide is

DDPKLYDKDLGSAMGDTVVGQMDAATSL (SEQ ID NO: 41)

Other methods to protect a cupredoxin derived peptide frombiotransformative degradation are N-acetylation and C-amidation. Thesederivatives may protect the peptide from degradation and may make thecupredoxin derived peptide more closely mimic the charge state of thealpha amino and carboxyl groups in the native protein. Peptides with theN-acetylation and/or C-amidation can be provided by commercialsuppliers. In one embodiment of the invention, the N-terminus of thecupredoxin derived peptide may be acetylated. In another embodiment ofthe invention, the C-terminus of the cupredoxin derived peptides may beamidated. In one specific embodiment, the modified cupredoxin derivedpeptide is

(SEQ ID NO: 42) Acetylation-LSTAADMQGVVTDGMASGLDKDYLKPDD-amidation

Cyclization is an additional manner of biotransformation that may bebeneficial to therapeutic peptides including the cupredoxins asdescribed herein. Cyclization may stabilize therapeutic peptides,allowing them to be stored longer, be administered at lower doses and beadministered less frequently. Cyclization has been shown to protectpeptides against peptidase and protease degradation. Cyclization can bedone chemically or enzymatically. Enzymatic cyclization is generallyless problematic than chemical cyclization, as chemical cyclization canlack in regio- and stereospecificity, can lead to multimerization inlieu of cyclization and can require complicated multistep processes.Indeed, it has been shown that thioether cyclization is more protectiveand stable than a disulfide bond against proteolytic enzymes.

Enzymatic cyclization has been shown inlantibiotics-(methyl)lanthionine-containing bacterial peptides. E.g., R.Rink, et al., “Lantibiotic Structures as Guidelines for the Design ofPeptides That Can Be Modified by Lantibioitic Enzymes” 44 Biochem.,8873-82 (2005); R. Rink, et al., “Production of DehydroaminoAcid-Containing Peptides by Lactococcus lactis” 73:6 Applied andEnvironmental Microbiology, 1792-96 (2007); R. Rink, et al., “NisC, theCyclase of the Lantibiotic Nisin, Can Catalyze Cyclization of DesignedNonlantibiotic Peptides” 46 Biochem., 13179-89 (2007) (each of which ishereby incorporated by reference in its entirety). Lantibiotics areproduced by and inhibit the growth of gram-positive bacteria. Inlantibiotics, dehydroalanine and dehydrobutyrine are created by enzymemediated dehydration of serine and threonine residues. Cysteines arethen enzymatically coupled to the dehydrated serine and threonineresidues to form thioether cyclizations. Naturally occurringlantibiotics show such couplings via thioether bonds between residuesthat are up to 19 residues apart. Thioether ring formation depends uponthe leader peptide. The location of the cyclization depends upon thecyclase mediated regio- and stereospecific ring closure and thepositions of the dehydratable serine and threonine residues.

The best characterized of the lantibiotics is nisin—a pentacyclicpeptide antiobiotic produced by Lactococcus lactis. Nisin is composed offour methyllanthionines, one lanthionine, two dehydroalanines, onedehydrobutyrine, and twenty-six unmodified amino acids. Nisin's fivethioether cross-links are formed by the addition of cysteine residues todehydroalanine and dehydrobutyrine residues that originate from serineand threonine. Nisin contains thioether-containing amino acids that areposttranslationally introduced by a membrane-associated enzyme complex.This enzyme complex includes: transporter NisT, serine and threoninedehydratase NisB, and cyclase NisC. NisB dehydrates serine and threonineresidues, converting them into dehydroalanine and dehydrobutyrine,respectively. This is followed by NisC catalyzed enantioselectivecoupling of cysteines to the formed dehydroresidues. NisT facilitatesthe export of the modified prenisin. Another enzyme, NisP cleaves thenisin leader peptide from prenisin.

The cyclase NisC has been well characterized. Li et al, “Structure andMechanism of the Lantibiotic Cyclase Involved in Nisin Biosynthesis” 311Science, 1464-67 (2006) (hereby incorporated by reference in itsentirety).

An analysis of cyclization in lantibiotics has led to the identificationof amino acid sequences and characteristics in peptides that favorcyclization. It has been shown that the NisB enzyme dehydrates moreoften where certain amino acids flank the serine and threonine residues.It has been shown that cyclization occurs more often in lantibioticpropeptides where hydrophobic, nonaromatic residues are in proximity tothe serine and threonine residues. The flanking residues of the modifiedcysteines are typically less hydrophobic than the flanking residues ofthe modified threonines and serines. Exceptions have been found,including hexapeptides VSPPAR (SEQ ID NO: 43), YTPPAL (SEQ ID NO: 44)and FSFFAF (SEQ ID NO: 45). The hexapeptides suggest that the presenceof a proline at position 3 or 4 or having phenylalanine flanking bothsides may prohibit dehydration. The rings are typically formed bycoupling a dehydrated residue to a C-terminally located cysteine.However, rings may be formed by coupling a dehydrate residue to aN-terminally located cysteine.

It has also been shown that the nisin dehydrating and transport enzymesare not specific to nisin and may, in fact, be used to modify non-nisinpeptides (and non-lantibiotic peptides). NisB has been shown todehydrate serine and threonine residues in peptides such as humanpeptide hormones when such peptides are N-terminally fused to thelantibiotic leader peptide. On non-lantibiotic peptides, similar ringformation characteristics apply; namely, the extent of dehydration canbe controlled by the amino acid context of the flanking region of thedehydratable serine and threonine residues. The presence of hydrophobicflanking residues (e.g., alanine and valine) around the serines andthreonines allowed full dehydration and therefore enhanced thioetherring formation. The presence of an N-terminal aspartate and C-terminallyflanked arginine prevented dehydration. It also shown that the presenceof proline residues and phenylalanine residues is disfavorable fordehydration. Generally, the presence of hydrophilic flanking residuesprevented dehydration of the serine and threonine residues. Hydrophobicflanking favors dehydration; hydrophilic flanking disfavors dehydration.Studies have shown that where dehydration does occur, the averagehydrophobicity of the flanking residues of serines and threonine ispositive—0.40 on the N-terminal side and 0.13 on the C-terminal side.Also, the average hydrophobicity of the residues flanking serines andthreonines that are not dehydrated is negative—−0.36 on the N-terminalside and −1.03 on the C-terminal side. Deydration is not restricted bythe presence of a series of flanking threonine residues and is notrestricted by the distance between the nisin leader peptide and theresidue to be dehydrated.

N is C has been shown to catalyze the regiospecific formation ofthioether rings in peptides unrelated to naturally occurringlantibiotics. Generally, such peptides must be fused to the nisin leaderpeptide. In some cases, thioether rings may form spontaneously, forexample where a dehydroalanine is spaced by two amino acids from acysteine. Unlike spontaneous cyclization, N is C catalyzed cyclizationis stereospecific for dehydrated pre-nisin. Consequently, themethyllanthionines and lanthionine in nisin are in the DL configuration.It is thought that cyclization in nonlantibiotic peptides will also bestereospecific

These principles can be applied to the compounds described herein,including cupredoxins and variants and truncations thereof.

Thioether Bridges

In nature, lantibiotic-enzyme-induced thioether bridges occur with up to19 amino acids under the bridge. Thioether bridges with 2 to 4 aminoacids under the bridge are abundant.

In some embodiments, the cupredoxin may be modified by introducingthioether bridges into the structure. The azurin truncation p28 (SEQ IDNO: 2), for example, may be modified using this method. Extendedmolecular dynamics simulations (70 ns) using software package GROMACS(www.gromacs.org) suggest that, at 37° C., the region of the p28 alphahelix from position 6 to 16 is unstable, and that the peptide tends toadopt a beta sheet conformation. FIG. 58, A and B. This, together withthe fact that the part of the molecule presumed to be responsible forinteraction with p53 remains solvent exposed, suggests that introductionof a thioether bridge in this region of the p28 peptide may not affectits functionality.

The amino acid sequence of p28 is SEQ ID NO: 2(LSTAADMQGVVTDGMASGLDKDYLKPDD). The amino acid sequence known as p18 isSEQ ID NO: 25 (LSTAADMQGVVTDGMASG). The sequence SGLDKD (SEQ ID NO: 96)may interact with p53. Thioether bridges can be formed between Ser/Thron the N-side to Cys on the C-side. The serine/threonine is dehydratedand subsequently coupled to the cysteine. Threonines are preferred sincethey are more easily dehydrated than serines. Generally, hydrophobicflanking residues (at least one) to the threonine are preferred sincethey enhance the extent of dehydration. Negatively charged amino acids,glutamate and aspartate, that are flanking residues have a strongnegative effect on dehydration. Generally, hydrophilic flankingresidues, especially glycin, do not favor dehydration. Preceding the Cysthere is a slight preference for charged hydrophilic residues,especially glutamate/aspartate. Depending on the size of the thioetherring, the bulkiness of the amino acids that participate in the ringmatters.

In one embodiment, the truncated azurin sequence isLSTAADMQGVVTDGMASGLDKDYLTPGC (SEQ ID NO: 46). A thioether bridge isformed between positions 25 and 28 of p28, and will be fully protectedagainst carboxyetidases. Positions 2, 3 and 25 will be dehydrated, butneither the import sequence, nor the sequence thought to be relevant forinteraction with p53, is altered by thioether ring introduction. Assuch, peptide activity should not be altered. The threonine is betweentwo hydrophobic amino acids and hence is expected to be fully dehydratedby dehydratase, NisB, according to specific guidelines. See Rink et al.,Biochemistry 2005. The same guidelines also predict cyclizationinvolving positions 25 and 28 by cyclase NisC, especially because of theaspartate located before the cysteine.

In another embodiment, the truncated azurin sequence isLSTAADCQGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 47) and the thioether bridge isformed between positions 3 and 7. The ring between position 3 and 7mimics ring A of nisin and makes use of the existing threonine atposition 2. The aspartate at position 6 will favor cyclization.

In another embodiment, the truncated azurin sequence isLSTAACMQGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 48), and the threonine inposition 2 is utilized to form a thioether bridge.

In another embodiment, two or more of the thioether rings in thetruncated azurins described in the paragraphs above are combined intoone peptide.

In another embodiment, many truncated azurin sequences can be createdand screened for threonine rings by analyzing the peptides with a ringof one lanthionine and two to three additional amino acids under thesulfur bridge. This might involve one or combinations of the sequencesbelow:

LSTACDMQGVVTDGMASGLDKDYLKPDD (SEQ ID NO: 49)LSTAATMQCVVTDGMASGLDKDYLKPDD (SEQ ID NO: 50)LSTAATMQGCVTDGMASGLDKDYLKPDD (SEQ ID NO: 51)LSTAANTQGCVTDGMASGLDKDYLKPDD (SEQ ID NO: 52)LSTAANTQGVCTDGMASGLDKDYLKPDD (SEQ ID NO: 53)LSTAADMTAVCTDGMASGLDKDYLKPDD (SEQ ID NO: 54)LSTAADMTAVVCDGMASGLDKDYLKPDD (SEQ ID NO: 55)LSTAADMQTVVCDGMASGLDKDYLKPDD (SEQ ID NO: 56)LSTAADMQTVVTCGMASGLDKDYLKPDD (SEQ ID NO: 57)LSTAADMQATVTCGMASGLDKDYLKPDD (SEQ ID NO: 58)LSTAADMQATVTDCMASGLDKDYLKPDD (SEQ ID NO: 59)LSTAADMQGVTADCMASGLDKDYLKPDD (SEQ ID NO: 60)LSTAADMQGVTADGCASGLDKDYLKPDD (SEQ ID NO: 61)LSTAADMQGVVTNGCASGLDKDYLKPDD (SEQ ID NO: 62)

A practical approach would be to genetically make a large number of suchsequences and select a group for purification on the basis of extent ofmodification and level of production.

In another embodiment, a thioether bridge is formed between a threonineat position 12 in p28 (SEQ ID NO: 2) and the c-terminus of the peptide.The distance between the Cα of position 13 and the aspartate at position28 might be 17.52 angstroms, larger than 1.5 nanometers, implyingsignificant alteration of the structure of the peptide. FIG. 58 C.

In another embodiment, the peptide sequence isLSTAADMQGVVTATMGSGLCKDYLKPDD (SEQ ID NO: 63), with a thioether bridgefrom position 14 to position 2 at a distance of 4.38 angstroms. Themutation of aspartate at position 13 to alanine favors dehydration ofthreonine at position 14. Mutation of alanine at position 16 to glycinecompletely prevents dehydration of serine at position 17 and enhancescyclization.

In another embodiment, the peptide sequence isLSTAADMQGVVTDLTASGLCKDYLKPDD (SEQ ID NO:64), with the thioether bridgefrom position 15 to position 20 at a distance of 5.83 angstroms. In thissituation, mutation of glycine at position 14 to leucine favorsdehydration of threonine at position 15.

Tertiary Structure Stabilization

The stability of the tertiary structure of the cupredoxin derivedpeptide will affect most aspects of the pharmacokinetics, including thepharmacologic activity, plasma half-life, and/or immunogenicity amongothers. See Kanovsky et al., Cancer Chemother. Pharmacol. 52:202-208(2003); Kanovsky et al., PNAS 23:12438-12443 (2001). Peptide helicesoften fall apart into random coils, becoming more susceptible toprotease attack and may not penetrate cell membrane well. Schafmeisteret al., J. Am. Chem. Soc. 122:5891-5892 (2000). Therefore, one way tostabilize the overall structure of the peptide is to stabilize theα-helix structure of the peptide. The intra-molecular hydrogen bondingassociated with helix formation reduces the exposure of the polar amidebackbone, thereby reducing the barrier to membrane penetration in atransport peptide, and thus increasing related pharmacologic activitiesand increasing the resistance of the peptide to protease cleavage. Id.Pseudomonas aeruginosa azurin (SEQ ID NO: 1) has α-helices at residues53-56, 58-64 and 68-70.

One method to stabilize an α-helix is to replace in the α-helix helixbreaking amino acid residues such as glycine, proline, serine andaspartic acid, or helix neutral amino acid residues such as alanine,threonine, valine, glutamine, asparagine, cysteine, histidine, lysine orarginine, with helix forming residues, such as leucine, isoleucine,phenylalanine, glutamic acid, tyrosine, tryptophan and methionine orhelix favoring amino acid residue substitutions, for exampleα-amino-isobutyric acid (Aib). See Miranda et al., J. Med. Chem., 51,2758-2765 (2008), the disclosure of which is incorporated by referenceherein. It is contemplated that the α-helix of cupredoxin derivedpeptides may be stabilized by replacing one or more glycine, proline,serine and/or aspartic acid residues with other amino acids. In specificembodiments, the glycine, proline, serine, aspartic acid, alanine,threonine, valine, glutamine, asparagine, cysteine, histidine, lysineand/or arginine residues are replaced by leucine, isoleucine,phenylalanine, glutamic acid, tyrosine, tryptophan, Aib and/ormethionine residues. See Lee et al., Cancer Cell Intl. 11:21 (2005). Inother specific embodiments, one or more serine or glutamine residues inthe α-helices of a cupredoxin derived peptide may be substituted. Instill more specific embodiments, the serine and/or glutamine residues inresidues 53-56, 58-64 and 68-70 of P. aeruginosa azurin (SEQ ID NO: 1),or equivalent residues of other cupredoxin derived peptides, may bereplaced. In another specific embodiment, the glutamine residue at aminoacid residue 57 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalentresidue of another cupredoxin derived peptide, may be replaced, morespecifically replaced with tryptophan. In another specific embodiment,the threonine residue at amino acid residue 52 of P. aeruginosa azurin(SEQ ID NO: 1), or an equivalent residue of another cupredoxin derivedpeptide, may be replaced, more specifically replaced with tryptophan. Inanother specific embodiment, the threonine residue at amino acid residue61 of P. aeruginosa azurin (SEQ ID NO: 1), or an equivalent residue ofanother cupredoxin derived peptide, may be replaced, more specificallyreplaced with tryptophan. In another specific embodiment, the glycineresidue at amino acid residue 63 of P. aeruginosa azurin (SEQ ID NO: 1),or an equivalent residue of another cupredoxin derived peptide, may bereplaced, more specifically replaced with tryptophan. In anotherspecific embodiment, one or more threonine, glutamine or glycineresidues at amino acid residues 52, 57, 61 or 63 of P. aeruginosa azurin(SEQ ID NO: 1), or an equivalent residue of another cupredoxin derivedpeptide, may be replaced, more specifically replaced with tryptophan. Inspecific embodiments, the cupredoxin peptide comprises one of thefollowing sequences wherein the underlined amino acid is substitutedinto the wildtype Pseudomonas aeruginosa p28 sequence:

LSWAADMQGVVTDGMASGLDKDYLKPDD; (SEQ ID NO: 65)LSTAADMWGVVTDGMASGLDKDYLKPDD; (SEQ ID NO: 66)LSTAADMQGVVWDGMASGLDKDYLKPDD; (SEQ ID NO: 67)LSTAADMQGVVTDWMASGLDKDYLKPDD; (SEQ ID NO: 68)LSWAADMWGVVTDGMASGLDKDYLKPDD; (SEQ ID NO: 69)LSWAADMQGVVWDGMASGLDKDYLKPDD; (SEQ ID NO: 70)LSWAADMQGVVTDWMASGLDKDYLKPDD; (SEQ ID NO: 71)LSTAADMWGVVWDGMASGLDKDYLKPDD; (SEQ ID NO: 72)LSTAADMWGVVTDWMASGLDKDYLKPDD; (SEQ ID NO: 73)LSTAADMQGVVWDWMASGLDKDYLKPDD; (SEQ ID NO: 74) orLSWAADMWGVVWDWMASGLDKDYLKPDD. (SEQ ID NO: 75)In other embodiments, equivalent amino acids in other cupredoxin derivedpeptides are substituted with tryptophan.

Another method to stabilize an α-helix tertiary structure involves usingunnatural amino acid residues capable of π-stacking. For example, inAndrews and Tabor (Tetrahedron 55:11711-11743 (1999)), pairs ofε-(3,5-dinitrobenzoyl)-Lys residues were substituted into the α-helixregion of a peptide at different spacings. The overall results showedthat the i,(i+4) spacing was the most effective stabilizing arrangement.Increasing the percentage of water, up to 90%, increased the helicalcontent of the peptide. Pairs of α-acyl-Lys residues in the same i,(i+4)spacing had no stabilizing effect, indicating that the majority of thestabilization arises from π-π interactions. In one embodiment, thecupredoxin derived peptide may be modified so that the lysine residuesare substituted by ε-(3,5-dinitrobenzoyl)-Lys residues. In a specificembodiment, the lysine residues may be substituted byε-(3,5-dinitrobenzoyl)-Lys in a i,(i+4) spacing.

Another method to stabilize an α-helix tertiary structure uses theelectrostatic interactions between side-chains in the α-helix. WhenHis-Cys or His-His residue pairs were substituted in into peptides in ani,(i+4) arrangement, the peptides changed from about 50% helical toabout 90% helical on the addition of Cu, Zn or Cd ions. When ruthenium(Ru) salts were added to the His-His peptides, an exchange-inert complexwas formed, a macrocyclic cis-[Ru—(NH₃)₄L₂]³⁺ complex where L₂ are theside chains of two histidines, which improved the helix stability.Ghadiri and Fernholz, J. Am. Chem. Soc. 112, 9633-9635 (1990). In someembodiments, the cupredoxin derived peptides may comprise macrocycliccis-[Ru—(NH₃)₄L₂]³⁺ complexes where L₂ is the side chains of twohistidines. In some embodiments, one or more histidine-cysteine orhistidine-histidine residue pairs may be substituted an i,(i+4)arrangement into the α-helices of the cupredoxin derived peptide. Inother embodiments, one or more histidine-cysteine or histidine-histidineresidue pairs may be substituted an i,(i+4) arrangement in residues53-56, 58-64 and 68-70 of P. aeruginosa azurin (SEQ ID NO: 1), orequivalent residues of other cupredoxin derived peptides. In someembodiments, the cupredoxin derived peptide may further comprise Cu, Zn,Cd and/or Ru ions.

Another method to stabilize an α-helix tertiary structure involvesdisulfide bond formation between side-chains of the α-helix. It is alsopossible to stabilize helical structures by means of formal covalentbonds between residues separated in the peptide sequence. The commonlyemployed natural method is to use disulfide bonds. Pierret et al., Intl.J. Pept. Prot. Res., 46:471-479 (1995). In some embodiments, one or morecysteine residue pairs are substituted into the α-helices of thecupredoxin derived peptide. In other embodiments, one or more cysteineresidue pairs are substituted at residues 53-56, 58-64 and 68-70 of P.aeruginosa azurin (SEQ ID NO: 1), or equivalent residues of othercupredoxin derived peptides.

Another method to stabilize an α-helical tertiary structure involves theuse of side chain lactam bridges. A lactam is a cyclic amide which canform from the cyclisation of amino acids. Side chain to side chainbridges have been successfully used as constraints in a variety ofpeptides and peptide analogues, such as amphipathic or model α-helicalpeptides, oxytocin antagonists, melanoptropin analogues, glucagon, andSDF-1 peptide analogues. For example, the Glucagon-like Peptide-1(GLP-1) gradually assumes a helical conformation under certainhelix-favoring conditions and can be stabilized using lactam bridging.Miranda et al., J. Med. Chem., 51, 2758-2765 (2008). These lactambridges may be varied in size, effecting stability and binding affinity.Id. Such modifications improved the stability of the compounds inplasma. Id. Depending on the space between the cyclization sites andchoice of residues, lactam bridges can be used to induce and stabilizeturn or helical conformations. In some embodiments, one or morecupredoxin or variant analogues are prepared with lactam bridgingbetween nearby amino acids (such as i to i+4 glutamic acid-lysineconstraints). In some embodiments, the cupredoxin derived peptide maycomprise such modifications to enhance α-helix content.

Another method to stabilize an α-helix tertiary structure is theall-carbon cross-link method. The all-hydrocarbon cross-link method isproven to increase the stabilization of helical structure, proteaseresistant and cell-permeability. Walensky et al., Science, 305,1466-1470 (2004). α,α-disubstituted non-natural amino acids containingolefin-bearing tethers are incorporated into peptides. Rutheniumcatalyzed olefin metathesis generates an all-hydrocarbon “staple” tocross-link the helix. Schafmeister et al., J. Am. Chem. Soc., 122,5891-5892 (2000); Walensky et al., id. Non-natural amino acidscontaining olefin-bearing tethers may be synthesized according tomethodology provided in Schafmeister et al. (id.) and Williams and Im(J. Am. Chem. Soc., 113:9276-9286 (1991)). In some embodiments, thecupredoxin derived peptides are stabilized by all-hydrocarbon staples.In specific embodiments, one or more pairs of α,α-disubstitutednon-natural amino acids containing olefin-bearing tethers correspondingto the native amino acids are substituted into the α-helices of thecupredoxin derived peptide. In other embodiments, one or more pairs ofα,α-disubstituted non-natural amino acids containing olefin-bearingtethers corresponded to the native amino acids are substituted intoresidues 53-56, 58-64 and 68-70 of P. aeruginosa azurin (SEQ ID NO: 1),or equivalent residues of other cupredoxin derived peptides.

In some embodiments, the modified cupredoxin derived peptide maycomprise X₁SX₂AADX₃X₄X₅VVX₆DX₇X₈ASGLDKDYLKPDX₉ (SEQ ID NO: 76), where X₁is L or acetylated-L, X₂ is T or W, X₃ is M, L or V, X₄ is Q or W, X₅ isG or A, X₆ is T or W, X₇ is G, T or W, X₈ is M, L or V, and X₉ is D oramidated-D. In other embodiments, the modified cupredoxin derivedpeptide may consist of X₁SX₂AADX₃X₄X₅VVX₆DX₇X₈ASGLDKDYLKPDX₉ (SEQ ID NO:76), where X₁ is L or acetylated-L, X₂ is T or W, X₃ is M, L or V, X₄ isQ or W, X₅ is G or A, X₆ is T or W, X₇ is G, T or W, X₈ is M, L or V,and X₉ is D or amidated-D.

In other embodiments, the modified cupredoxin derived peptide maycomprise X₁DPKLYDKDLGSAX₂X₃DX₄VVX₅X₆X₇DAAX₈SX₉ (SEQ ID NO: 77), where X₁is D or acetylated-D, X₂ is M, L or V, X₃ is G, T or W, X₄ is T or W, X₅is G or A, X₆ is Q or W, X₇ is M, L or V, X₈ is T or W, and X₉ is L oramidated-L. In other embodiments, the modified cupredoxin derivedpeptide may consist of X₁DPKLYDKDLGSAX₂X₃DX₄VVX₅X₆X₇DAAX₈SX₉ (SEQ ID NO:77), where X₁ is D or acetylated-D, X₂ is M, L or V, X₃ is G, T or W, X₄is T or W, X₅ is G or A, X₆ is Q or W, X₇ is M, L or V, X₈ is T or W,and X₉ is L or amidated-L. Specific peptides of interest are listed inTable 3.

PEGylation

Covalent attachment of PEG to drugs of therapeutic and diagnosticimportance has extended the plasma half-life of the drug in vivo, and/orreduced their immunogenicity and antigenicity. Harris and Chess, NatureReviews Drug Discovery 2:214-221 (2003). For example, PEG attachment hasimproved the pharmacokinetic properties of many therapeutic proteins,including interleukins (Kaufman et al., J. Biol. Chem. 263:15064 (1988);Tsutsumi et al., J. Controlled Release 33:447 (1995)), interferons (Kitaet al., Drug Des. Delivery 6:157 (1990)), catalase (Abuchowski et al.,J. Biol. Chem. 252:3582 (1977)), superoxide dismutase (Beauchamp et al.,Anal. Biochem. 131:25 (1983)), and adenosine deanimase (Chen et al.,Biochem. Biophys. Acta 660:293 (1981)), among others. The FDA hasapproved PEG for use as a vehicle or base in foods, cosmetics andpharmaceuticals, including injectable, topical, rectal and nasalformulations. PEG shows little toxicity, and is eliminated from the bodyintact by either the kidneys (for PEGs <30 kDa) or in the feces (forPEGs >20 kDa). PEG is highly soluble in water.

PEGylation of a therapeutic peptide may be used to increase the lifetimeof the peptide in the bloodstream of the patient by reducing renalultrafiltration, and thus reduce elimination of the drug from the body.Charge masking may affect renal permeation. Charge masking may be aconsequence of the paramchemical modification of protein ionizablefunctional group, namely amines or carboxyls. In particular, the mostcommon procedures for producing protein-PEG derivatives involves theconversion of protein amino groups into amides with the consequent lossof positive charges, and this can alter protein ultrafiltration. Sinceanionic macromolecules have been found to be cleared by renalultrafiltration more slowly than neutral or positive ones, it could beexpected that PEG conjugation to amino groups prolongs the permanence ofthe PEGylated peptide in the bloodstream.

Molecular size and globular ultrafiltration may also affect renalultrafiltration of therapeutic peptides. The molecular weight cut offfor kidney elimination of native globular proteins is considered to beabout 70 kDa, which is close to the molecular weight of serum albumin.Thus, proteins with molecular weight exceeding 70 kDa are mainlyeliminated from the body by pathways other than renal ultrafiltration,such as liver uptake, proteolytic digestion and clearance by the immunesystem. Therefore, increasing the size of a therapeutic peptide byPEGylation may decrease renal ultrafiltration of that peptide form thebloodstream of the patient.

Additionally, PEGylation of a therapeutic peptide may decrease theimmunogenicity of that peptide, as well as protect the peptide fromproteolytic enzymes, phagocytic cells, and other factors that requiredirect contact with the therapeutic peptide. The umbrella-like structureof branched PEG in particular has been found to give better protectionthan linear PEG towards approaching proteolytic enzymes, antibodies,phagocytic cells, etc. Caliceti and Veronese, Adv. Drug. Deliv. Rev.55:1261-12778 (2003).

In some embodiments, the cupredoxin derived peptides of the inventionare modified to have one or more PEG molecules covalently bonded to acysteine molecule. The covalent bonding does not necessarily need to bea covalent bond directly from the PEG molecule to the cupredoxin derivedpeptide, but may be covalently bonded to one or more linker moleculeswhich in turn are covalently bonded to each other and/or the cupredoxinderived peptide. In some embodiments, the cupredoxin derived peptidehave site-specific PEGylation. In specific embodiments, the PEGmolecule(s) may be covalently bonded to the cysteine residues 3, 26and/or 112 of P. aeruginosa azurin (SEQ ID NO: 1). In other embodiments,one or more cysteine residues may be substituted into the cupredoxinderived peptide and is PEGylated. In some embodiments, the method toPEGylate the cupredoxin derived peptide may be NHS, reductive animation,malimid or epoxid, among others. In other embodiments, the cupredoxinderived peptides may be PEGylated on one or more lysine, cysteine,histidine, arginine, aspartic acid, glutamic acid, serine, threonine, ortyrosine, or the N-terminal amino group or the C-terminal carboxylicacid. In more specific embodiments, the cupredoxin derived peptides maybe PEGylated on one or more lysines or N-terminal amino groups. In otherembodiments, one or more lysine, cysteine, histidine, arginine, asparticacid, glutamic acid, serine, threonine, or tyrosine residue aresubstituted into the cupredoxin derived peptides and are PEGylated. Inother embodiments, the cupredoxin derived peptides may be PEGylated onone or more amino groups. In other embodiments, the cupredoxin derivedpeptides may be PEGylated in a random, non-site specific manner. In someembodiments, the cupredoxin derived peptides may have an averagemolecular weight of PEG-based polymers of about 200 daltons to about100,000 daltons, about 2,000 daltons to about 20,000 daltons, or about2,000 daltons to about 5,000 daltons. In other embodiments, thecupredoxin derived peptides may be comprised of one or more PEGmolecules that is branched, specifically a branched PEG molecule that isabout 50 kDa. In other embodiments, the cupredoxin derived peptides maycomprise one or more linear PEG molecules, specifically a linear PEGmolecule that is about 5 kDa.

In another embodiment, the peptide is a cupredoxin, or variant,structural equivalent, or derivative thereof that is a conjugate ofPep42, a cyclic 13-mer oligopeptide that specifically binds toglucose-regulated protein 78 (GRP78) and is internalized into cancercells. The cupredoxin or variant, structural equivalent, or derivativeof cupredoxin may be conjugated with Pep42 pursuant to the synthesismethods disclosed in Yoneda et al., “A cell-penetrating peptidic GRP78ligand for tumor cell-specific prodrug therapy,” Bioorganic & MedicinalChemistry Letters 18: 1632-1636 (2008), the disclosure of which isincorporated in its entirety herein.

In another embodiment, the peptide is a structural equivalent of acupredoxin. Examples of studies that determine significant structuralhomology between cupredoxins and other proteins include Toth et al.(Developmental Cell 1:82-92 (2001)). Specifically, significantstructural homology between a cupredoxin and the structural equivalentmay be determined by using the VAST algorithm. Gibrat et al., Curr OpinStruct Biol 6:377-385 (1996); Madej et al., Proteins 23:356-3690 (1995).In specific embodiments, the VAST p value from a structural comparisonof a cupredoxin to the structural equivalent may be less than about10⁻³, less than about 10⁻⁵, or less than about 10⁻⁷. In otherembodiments, significant structural homology between a cupredoxin andthe structural equivalent may be determined by using the DALI algorithm.Holm & Sander, J. Mol. Biol. 233:123-138 (1993). In specificembodiments, the DALI Z score for a pairwise structural comparison is atleast about 3.5, at least about 7.0, or at least about 10.0.

It is contemplated that the peptides of the composition of invention maybe more than one of a variant, derivative, truncation and/or structuralequivalent of a cupredoxin. For example, the peptides may be atruncation of azurin that has been PEGylated, thus making it both atruncation and a derivative. In one embodiment, the peptides of theinvention are synthesized with α,α-disubstituted non-natural amino acidscontaining olefin-bearing tethers, followed by an all-hydrocarbon“staple” by ruthenium catalyzed olefin metathesis. Scharmeister et al.,J. Am. Chem. Soc. 122:5891-5892 (2000); Walensky et al., Science305:1466-1470 (2004). Additionally, peptides that are structuralequivalents of azurin may be fused to other peptides, thus making apeptide that is both a structural equivalent and a derivative. Theseexamples are merely to illustrate and not to limit the invention.Variants, derivatives or structural equivalents of cupredoxin may or maynot bind copper.

In some embodiments, the cupredoxin, or variant, derivative orstructural equivalent thereof has some of the pharmacologic activitiesof the P. aeruginosa azurin, and specifically p28. In a specificembodiment, the cupredoxins and variants, derivatives and structuralequivalents of cupredoxins that may inhibit prevent the development ofpremalignant lesions in mammalian cells, tissues or animals, andspecifically but not limited to, mammary gland cells. The invention alsoprovides for the cupredoxins and variants, derivatives and structuralequivalents of cupredoxins that may have the ability to inhibit thedevelopment of mammalian premalignant lesions, and specifically but notlimited to, melanoma, breast, pancreas, glioblastoma, astrocytoma, lung,colorectal, neck and head, bladder, prostate, skin and cervical cancercells. Inhibition of the development of cancer cells is any decrease, orlessening of the rate of increase, of the development of premalignantlesions that is statistically significant as compared to controltreatments.

Because it is now known that cupredoxins can preferentially enter cancercells via endocytotic pathways, and can also inhibit the development ofpremalignant lesions and ultimately cancer in mammalian cells, tissuesor animals, and specifically breast cells, and more specifically, mousemammary gland cells, it is now possible to design variants andderivatives of cupredoxins that retain this activity. Such variants,derivatives and structural equivalents can be made by, for example,creating a “library” of various variants, derivatives and structuralequivalents of cupredoxins and cupredoxin derived peptides and thentesting each for preferential entry and/or chemopreventive activity, andspecifically preferential entry and/or chemopreventive activity in themouse mammary gland organ culture using one of many methods known in theart, such the exemplary method in Example 1. It is contemplated that theresulting variants, derivatives and structural equivalents ofcupredoxins with chemopreventive activity and/or the ability topreferentially enter cells may be used in the methods of the invention,in place of or in addition to azurin or p28.

In some specific embodiments, the variant, derivative or structuralequivalent of cupredoxin may inhibit the development of7,12-dimethylbenz (a) anthracene (DMBA) induced premalignant lesions ina mouse mammary gland organ culture (MMOC) to a degree that isstatistically different from a non-treated control. A peptide can betested for this activity by using the MMOC model system is described inExample 1, or as in Mehta et al. (J Natl Cancer Inst 93:1103-1106(2001)) and Mehta et al. (Meth Cell Sci 19:19-24 (1997)). Other methodsto determine whether cancer development is inhibited another are wellknown in the art and may be used as well.

In some specific embodiments, the variant, derivative or structuralequivalent of cupredoxin inhibits the development of mammary alveolarlesions (MAL) in the a MMOC model to a degree that is statisticallydifferent from a non-treated control. In some specific embodiments, thevariant, derivative or structural equivalent of cupredoxin inhibits thedevelopment of mammary ductal lesions (MDL) in the a MMOC model to adegree that is statistically different from a non-treated control. Apeptide can be tested for these activities by using the MMOC modelsystem induced to form premalignant lesions by DMBA, as described inExample 1. Evaluation of development of premalignant lesions in a MMOCmodel system may be determined by morphometic analysis, orhistopathological analysis, as provided in Example 1.

In some specific embodiments, the variant, derivative or structuralequivalent can preferentially enter cancer cells and/or tumors inmammalian cells, tissues and animals. In some embodiments, the variantis a derivative or structural equivalent of p18. In some embodiments,the variant, derivative or structural equivalent can selectively entercancer cells and/or tumors in mammalian cells, tissues and animals anddeliver DNA or RNA. In some embodiments, the DNA or RNA is a gene or aportion of a gene. In some embodiments, the DNA or RNA has a therapeuticeffect once delivered. In some embodiments, the variant is a derivativeor structural equivalent of p28. In some embodiments, the variant,derivative or structural equivalent can selectively enter cancer cellsand/or tumors in mammalian cells, tissues and animals and deliver DNA orRNA. In some embodiments, the DNA or RNA is a gene or a portion of agene. In some embodiments, the DNA or RNA has a therapeutic effect oncedelivered.

Cupredoxins

These small blue copper proteins (cupredoxins) are electron transferproteins (10-20 kDa) that participate in bacterial electron transferchains or are of unknown function. The copper ion is solely bound by theprotein matrix. A special distorted trigonal planar arrangement to twohistidine and one cysteine ligands around the copper gives rise to verypeculiar electronic properties of the metal site and an intense bluecolor. A number of cupredoxins have been crystallographicallycharacterized at medium to high resolution.

The cupredoxins in general have a low sequence homology but highstructural homology. Gough & Clothia, Structure 12:917-925 (2004); DeRienzo et al., Protein Science 9:1439-1454 (2000). For example, theamino acid sequence of azurin is 31% identical to that of auracyanin B,16.3% to that of rusticyanin, 20.3% to that of plastocyanin, and 17.3%to that of pseudoazurin. See, Table 1. However, the structuralsimilarity of these proteins is more pronounced. The VAST p value forthe comparison of the structure of azurin to auracyanin B is 10^(−7.4),azurin to rusticyanin is 10⁻⁵, azurin to plastocyanin is 10^(−5.6), andazurin to psuedoazurin is 10^(−4.1).

All of the cupredoxins possess an eight-stranded Greek key beta-barrelor beta-sandwich fold and have a highly conserved site architecture. DeRienzo et al., Protein Science 9:1439-1454 (2000). A prominenthydrophobic patch, due to the presence of many long chain aliphaticresidues such as methionines and leucines, is present around the coppersite in azurins, amicyanins, cyanobacterial plastocyanins, cucumberbasic protein and to a lesser extent, pseudoazurin and eukaryoticplastocyanins. Id. Hydrophobic patches are also found to a lesser extentin stellacyanin and rusticyanin copper sites, but have differentfeatures. Id.

TABLE 1 Sequence and structure alignment of azurin (1JZG) from P.aeruginosa to other proteins using VAST algorithm. Alignment % aa PDBlength¹ identity P-value² Score³ RMSD⁴ Description 1AOZ A 2 82 18.310e−7 12.2 1.9 Ascorbate oxidase 1QHQ_A 113 31 10e−7.4 12.1 1.9AuracyaninB 1V54 B 1 79 20.3 10e−6.0 11.2 2.1 Cytocrome c oxidase 1GY2 A92 16.3 10e−5.0 11.1 1.8 Rusticyanin 3MSP A 74 8.1 10e−6.7 10.9 2.5Motile Major Sperm Protein⁵ 1IUZ 74 20.3 10e−5.6 10.3 2.3 Plastocyanin1KGY E 90 5.6 10e−4.6 10.1 3.4 Ephrinb2 1PMY 75 17.3 10e−4.1 9.8 2.3Pseudoazurin ¹Aligned Length: The number of equivalent pairs of C-alphaatoms superimposed between the two structures, i.e. how many residueshave been used to calculate the 3D superposition. ²P-VAL: The VAST pvalue is a measure of the significance of the comparison, expressed as aprobability. For example, if the p value is 0.001, then the odds are1000 to 1 against seeing a match of this quality by pure chance. The pvalue from VAST is adjusted for the effects of multiple comparisonsusing the assumption that there are 500 independent and unrelated typesof domains in the MMDB database. The p value shown thus corresponds tothe p value for the pairwise comparison of each domain pair, divided by500. ³Score: The VAST structure-similarity score. This number is relatedto the number of secondary structure elements superimposed and thequality of that superposition. Higher VAST scores correlate with highersimilarity. ⁴RMSD: The root mean square superposition residual inAngstroms. This number is calculated after optimal superposition of twostructures, as the square root of the mean square distances betweenequivalent C-alpha atoms. Note that the RMSD value scales with theextent of the structural alignments and that this size must be takeninto consideration when using RMSD as a descriptor of overall structuralsimilarity. ⁵ C. elegans major sperm protein proved to be an ephrinantagonist in ocyte maturation. Kuwabara, Genes and Development 17:155-161 (2003).

Azurin

The azurins are copper containing proteins of 128 amino acid residueswhich belong to the family of cupredoxins involved in electron transferin certain bacteria. The azurins include those from P. aeruginosa (PA)(SEQ ID NO: 1), A. xylosoxidans, and A. denitrficans. Murphy et al., J.Mol. Biol. 315:859-871 (2002). The amino acid sequence identity betweenthe azurins varies between 60-90%, these proteins showed a strongstructural homology. All azurins have a characteristic β-sandwich withGreek key motif and the single copper atom is always placed at the sameregion of the protein. In addition, azurins possess an essentiallyneutral hydrophobic patch surrounding the copper site. Id.

Plastocyanins

The plastocyanins are soluble proteins of cyanobacteria, algae andplants that contain one molecule of copper per molecule and are blue intheir oxidized form. They occur in the chloroplast, where they functionas electron carriers. Since the determination of the structure of poplarplastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha,Chlamydomonas) and plant (French bean) plastocyanins has been determinedeither by crystallographic or NMR methods, and the poplar structure hasbeen refined to 1.33 Å resolution. SEQ ID NO: 3 shows the amino acidsequence of plastocyanin from Phormidium laminosum, a thermophiliccyanobacterium. Another plastocyanin of interest is from Ulva pertussis.

Despite the sequence divergence among plastocyanins of algae andvascular plants (e.g., 62% sequence identity between the Chlamydomonasand poplar proteins), the three-dimensional structures are conserved(e.g., 0.76 Å rms deviation in the C alpha positions between theChlamydomonas and Poplar proteins). Structural features include adistorted tetrahedral copper binding site at one end of aneight-stranded antiparallel beta-barrel, a pronounced negative patch,and a flat hydrophobic surface. The copper site is optimized for itselectron transfer function, and the negative and hydrophobic patches areproposed to be involved in recognition of physiological reactionpartners. Chemical modification, cross-linking, and site-directedmutagenesis experiments have confirmed the importance of the negativeand hydrophobic patches in binding interactions with cytochrome f, andvalidated the model of two functionally significant electron transferpaths involving plastocyanin. One putative electron transfer path isrelatively short (approximately 4 Å) and involves the solvent-exposedcopper ligand His-87 in the hydrophobic patch, while the other is morelengthy (approximately 12-15 Å) and involves the nearly conservedresidue Tyr-83 in the negative patch. Redinbo et al., J. Bioenerg.Biomembr. 26:49-66 (1994).

Rusticyanins

Rusticyanins are blue-copper containing single-chain polypeptidesobtained from a Thiobacillus (now called Acidithiobacillus). The X-raycrystal structure of the oxidized form of the extremely stable andhighly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans(SEQ ID NO: 4) has been determined by multiwavelength anomalousdiffraction and refined to 1.9 Å resolution. The rusticyanins arecomposed of a core beta-sandwich fold composed of a six- and aseven-stranded b-sheet. Like other cupredoxins, the copper ion iscoordinated by a cluster of four conserved residues (His 85, Cys138,His143, Met148) arranged in a distorted tetrahedron. Walter, R. L. etal., J. Mol. Biol. 263:730-51 (1996).

Pseudoazurins

The pseudoazurins are a family of blue-copper containing single-chainpolypeptide. The amino acid sequence of pseudoazurin obtained fromAchromobacter cycloclastes is shown in SEQ ID NO: 5. The X-ray structureanalysis of pseudoazurin shows that it has a similar structure to theazurins although there is low sequence homology between these proteins.Two main differences exist between the overall structure of thepseudoazurins and azurins. There is a carboxy terminus extension in thepseudoazurins, relative to the azurins, consisting of two alpha-helices.In the mid-peptide region azurins contain an extended loop, shortened inthe pseudoazurins, which forms a flap containing a short α-helix. Theonly major differences at the copper atom site are the conformation ofthe MET side-chain and the Met-S copper bond length, which issignificantly shorter in pseudoazurin than in azurin.

Phytocyanins

The proteins identifiable as phytocyanins include, but are not limitedto, cucumber basic protein, stellacyanin, mavicyanin, umecyanin, acucumber peeling cupredoxin, a putative blue copper protein in pea pods,and a blue copper protein from Arabidopsis thaliana. In all exceptcucumber basic protein and the pea-pod protein, the axial methionineligand normally found at blue copper sites is replaced by glutamine.

Auracyanin

Three small blue copper proteins designated auracyanin A, auracyaninB-1, and auracyanin B-2 have been isolated from the thermophilic greengliding photosynthetic bacterium Chloroflexus aurantiacus. The two Bforms are glycoproteins and have almost identical properties to eachother, but are distinct from the A form. The sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrates apparent monomermolecular masses as 14 (A), 18 (B-2), and 22 (B-1) kDa.

The amino acid sequence of auracyanin A has been determined and showedauracyanin A to be a polypeptide of 139 residues. Van Dreissche et al.,Protein Science 8:947-957 (1999). His58, Cys123, His128, and Met132 arespaced in a way to be expected if they are the evolutionary conservedmetal ligands as in the known small copper proteins plastocyanin andazurin. Secondary structure prediction also indicates that auracyaninhas a general beta-barrel structure similar to that of azurin fromPseudomonas aeruginosa and plastocyanin from poplar leaves. However,auracyanin appears to have sequence characteristics of both small copperprotein sequence classes. The overall similarity with a consensussequence of azurin is roughly the same as that with a consensus sequenceof plastocyanin, namely 30.5%. The N-terminal sequence region 1-18 ofauracyanin is remarkably rich in glycine and hydroxy amino acids. Id.See exemplary amino acid sequence SEQ ID NO: 15 for chain A ofauracyanin from Chloroflexus aurantiacus (NCBI Protein Data BankAccession No. AAM12874).

The auracyanin B molecule has a standard cupredoxin fold. The crystalstructure of auracyanin B from Chloroflexus aurantiacus has beenstudied. Bond et al., J. Mol. Biol. 306:47-67 (2001). With the exceptionof an additional N-terminal strand, the molecule is very similar to thatof the bacterial cupredoxin, azurin. As in other cupredoxins, one of theCu ligands lies on strand 4 of the polypeptide, and the other three liealong a large loop between strands 7 and 8. The Cu site geometry isdiscussed with reference to the amino acid spacing between the latterthree ligands. The crystallographically characterized Cu-binding domainof auracyanin B is probably tethered to the periplasmic side of thecytoplasmic membrane by an N-terminal tail that exhibits significantsequence identity with known tethers in several othermembrane-associated electron-transfer proteins. The amino acid sequencesof the B forms are presented in McManus et al. J. Biol. Chem.267:6531-6540 (1992). See exemplary amino acid sequence SEQ ID NO: 16for chain B of auracyanin from Chloroflexus aurantiacus (NCBI ProteinData Bank Accession No. 1QHQA).

Stellacyanin

Stellacyanins are a subclass of phytocyanins, a ubiquitous family ofplant cupredoxins. An exemplary sequence of a stellacyanin is includedherein as SEQ ID NO: 14. The crystal structure of umecyanin, astellacyanin from horseradish root (Koch et al., J. Am. Chem. Soc.127:158-166 (2005)) and cucumber stellacyanin (Hart et al., ProteinScience 5:2175-2183 (1996)) is also known. The protein has an overallfold similar to the other phytocyanins. The ephrin B2 protein ectodomaintertiary structure bears a significant similarity to stellacyanin. Tothet al., Developmental Cell 1:83-92 (2001). An exemplary amino acidsequence of a stellacyanin is found in the National Center forBiotechnology Information Protein Data Bank as Accession No. 1JER, SEQID NO: 14.

Cucumber Basic Protein

An exemplary amino acid sequence from a cucumber basic protein isincluded herein as SEQ ID NO: 17. The crystal structure of the cucumberbasic protein (CBP), a type 1 blue copper protein, has been refined at1.8 Å resolution. The molecule resembles other blue copper proteins inhaving a Greek key beta-barrel structure, except that the barrel is openon one side and is better described as a “beta-sandwich” or “beta-taco”.Guss et al., J. Mol. Biol. 262:686-705 (1996). The ephrinB2 proteinectodomian tertiary structure bears a high similarity (rms deviation 1.5Å for the 50 α carbons) to the cucumber basic protein. Toth et al.,Developmental Cell 1:83-92 (2001).

The Cu atom has the normal blue copper NNSS′ co-ordination with bondlengths Cu—N(His39)=1.93 A, Cu—S(Cys79)=2.16 A, Cu—N(His84)=1.95 A,Cu—S(Met89)=2.61 A. A disulphide link, (Cys52)-S—S-(Cys85), appears toplay an important role in stabilizing the molecular structure. Thepolypeptide fold is typical of a sub-family of blue copper proteins(phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3,with which CBP has a high degree of sequence identity. The proteinscurrently identifiable as phytocyanins are CBP, stellacyanin,mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative bluecopper protein in pea pods, and a blue copper protein from Arabidopsisthaliana. In all except CBP and the pea-pod protein, the axialmethionine ligand normally found at blue copper sites is replaced byglutamine. An exemplary sequence for cucumber basic protein is found inNCBI Protein Data Bank Accession No. 2CBP, SEQ ID NO: 17.

Methods of Use

The invention provides methods to prevent malignancies in otherwisehealthy patients comprising administering to the patient at least onepeptide that is a cupredoxin, or variant, derivative or structuralequivalent thereof, as described above. Chemopreventive therapies arebased on the hypothesis that the interruption of processes involved incarcinogenesis will prevent the development of cancer. The cupredoxinPseudomonas aeruginosa azurin and the truncated azurin peptide p28 arenow known to inhibit the development of premalignant lesions, either byinhibiting the initial formation of premalignant lesions, or killing orinhibiting the growth of premalignant lesions that are present.

It therefore contemplated that a cupredoxin, or variant, truncation,derivative or structural equivalent thereof, as described above, withthe ability to inhibit the development of premalignant lesions, may beused in chemopreventive therapies in otherwise healthy patients. Suchotherwise healthy patients are, in some embodiments, patients at ahigher risk to develop cancer than those in the general population.Cancers that may be prevented by treatment with the compositions of theinvention include, but are not limited to, melanoma, breast, pancreas,glioblastoma, astrocytoma, lung, colorectal, neck and head, bladder,prostate, skin, and cervical cancer. In some embodiments, the patientmay be human. In other embodiments, the patient is not human.

The invention further includes compositions and methods topreferentially enter cancerous cells. Cupredoxins, specifically azurinderivatives p18 and p28, are now known to enter cancerous cells viacertain mechanisms described herein, including caveolae-mediatedendocytosis, which may be mediated by the Golgi apparatus. It istherefore contemplated that a cupredoxin or variant, derivative, orstructural equivalent thereof may be used to enter and kill cancercells, and may also be used to transport cargo across cell membranes.

The invention further includes methods to study the development ofcancer comprising contacting mammalian cells before or after inductionwith a carcinogen with a composition comprising cupredoxin, or avariant, derivative, truncation, or structural equivalent thereof andobserving the development of the cells. In some embodiments, the cellsare mouse mammary gland cells, while in others they are other cells thatmay become malignant in mammals.

Patients at a higher at risk to develop cancer than the generalpopulation may be patients with high risk features, patients withpremalignant lesions, and patients that have been cured of their initialcancer or definitively treated for their premalignant lesions. Seegenerally Tsao et al., CA Cancer J Clin 54:150-180 (2004). High riskfeatures may be behavioral, genetic, environmental or physiologicalfactors of the patient. Behavioral factors that predispose a patient tovarious forms of cancer include, but are not limited to, smoking, diet,alcohol consumption, hormone replacement therapy, higher body massindex, nulliparity, beta1 nut use, frequent mouthwash use, exposure tohuman papillomavirus, childhood and chronic sun exposure, early age offirst intercourse, multiple sexual partners, and oral contraceptive use.Genetic factors that predispose a patient to various forms of cancerinclude, but are not limited to, a family history of cancer, genecarrier status of BRCA1 and BRCA2, prior history of breast neoplasia,familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectalcancer (HNPCC), red or blond hair and fair-skinned phenotype, xerodermapigmentosum, and ethnicity. Environmental features that predispose apatient to various forms of cancer include, but are not limited to,exposure to radon, polycyclic aromatic hydrocarbons, nickel, chromate,arsenic, asbestos, chloromethyl ethers, benzo[a]pyrene, radiation, andaromatic amines from rubber or paint occupational exposure. Othermiscellaneous factors that predispose a patient to various forms ofcancer include, but are not limited to, chronic obstructive pulmonarydisease with airflow obstruction, chronic bladder infections,schistosomiasis, older age, and immunocompromised status.

Additionally, patients at a higher risk of developing cancer may bedetermined by the use of various risk models that have been developedfor certain kinds of cancer. For example, patients predisposed to breastcancer may be determined using the Gail risk model, or the Claus model,among others. See Gail et al., J Natl Cancer Inst 81:1879-1886 (1989);Cuzick, Breast 12:405-411 (2003); Huang et al., Am J Epidemiol151:703-714 (2000).

Patients with premalignant lesions are at a higher risk to developcancer than the general population. The presence of premalignant lesionsin or on a patient may be determined by many methods that are well knownto those in the art. Intermediate markers or biomarkers that originatefrom premalignant lesions may be measured in a patient to determine ifthe patient harbors premalignant lesions. Chromosomal abnormalitiesoccur in tumor cells and the adjacent histologically normal tissues inthe majority of cancer patients. Progression in chromosomalabnormalities parallels the phenotypic progression from premalignantlesion to invasive cancer. Thiberville et al., Cancer Res. 55:5133-5139(1995). Therefore, chromosomal abnormalities associated with cancer maybe used as intermediate markers to detect premalignant lesions in apatient. Common chromosomal abnormalities associated with cancerinclude, but are not limited to, allelic deletions or loss ofheterozygosity (LOH) in tumor suppressor genes such as 3p (FHIT andothers), 9p (9p21 for p16^(INK4), p15^(INK4B), and p19^(ARF)), 17p(17p13 for p53 gene and others) and 13q (13q14 for retinoblastoma geneRb and others). Deletions in 3p and 9p are associated with smoking andthe early stages of lung cancer. Mao et al., J. Natl. Cancer Inst.89:857-862 (1997). Deletions affecting 3p, 5q, 8p, 17p and 18q arecommon change in epithelial cancers. See generally Tsao et al., CA Clin.Cancer J. Clin. 54:153 (2004). Other chromosomal mutations associatedwith cancer include those which activate oncogenes. Oncogenes whosepresence may be used as intermediate markers include, but are notlimited to, Ras, c-myc, epidermal growth factor, erb-B2 and cyclins E,D1 and B1. See generally id. at 154.

Other intermediate markers may be the products of genes up-regulated inpremalignant cells and cancer cells. Genes that may be up-regulated inpremalignant cells include, but are not limited to, cyclooxygenasesCOX-1 and COX-2, telomerase. Other biomarkers of cancer cells, and somepremalignant cells, include, but are not limited to, p53, epidermalgrowth factor receptor (GFR), proliferating cell nuclear antigen (PCNA),RAS, COX-2, Ki-67, DNA aneuploidy, DNA polymerase-α, ER, Her2neu,E-cadherin, RARβ, hTERT, p16^(INK4a), FHIT (3p14), Bcl-2, VEGF-R, HPVinfection, LOH 9p21, LOH 17p, p-AKT, hnRNP A2/B1, RAF, Myc, c-KIT,cyclin D1, E and B1, IGF1, bcl-2, p16, LOH 3p21.3, LOH 3p25, LOH 9p21,LOH 17p13, LOH 13q, LOH 8p, hMSH2, APC, DCC, DPC4, JV18, BAX, PSA,GSTP1, NF-kB, AP1, D3S2, HPV infection, LOH 3p14, LOH 4q, LOH 5p,bladder tumor antigen (BTA), BTK TRAK (Alidex, Inc., Redmond Wash.),urinary tract matrix protein 22, fibrin degradation product, autodrinemotility factor receptor, BCLA-4, cytokeratin 20, hyaluronic acid, CYFRA21-1, BCA, beta-human chorionic gonadotropin, and tissue polypeptideantigen (TPA). See generally id. at 155-157.

Patients that have been cured of their initial cancers or have beendefinitively treated for their premalignant lesions are also at a higherrisk to develop cancer than the general population. A second primarytumor refers to a new primary cancer in a person with a history ofcancer. Second primary tumors are the leading cause of mortality in headand neck cancer Id. at 150. A second primary tumor is distinct from ametastasis in that the former originates de novo while the lateroriginates from an existing tumor. Patients that have been cured ofcancer or premalignant lesions of the breast, head and neck, lung, andskin are at a particularly high risk to develop second primary tumors.

The compositions comprising a cupredoxin or variant, derivative,truncation, or structural equivalent thereof can be administered to thepatient by many routes and in many regimens that will be well known tothose in the art. In specific embodiments, the cupredoxin, or variant,derivative or structural equivalent thereof is administeredintravenously, intramuscularly, subcutaneously, topically, orally, or byinhalation. The compositions may be administered to the patient by anymeans that delivers the peptides to the site in the patient that is atrisk of developing cancer. In specific embodiments, the cupredoxin orvariant, derivative, truncation, or structural equivalent thereof isadministered intravenously.

In one embodiment, the methods may comprise co-administering to apatient one unit dose of a composition comprising a cupredoxin or avariant, derivative, truncation, or structural equivalent of cupredoxinand one unit dose of a composition comprising another chemopreventivedrug, in either order, administered at about the same time, or withinabout a given time following the administration of the other, forexample, about one minute to about 60 minutes following theadministration of the other drug, or about 1 hour to about 12 hoursfollowing the administration of the other drug. Chemopreventive drugs ofinterest include, but are not limited to, Tamoxifen, aromataseinhibitors such as letrozole and anastrozole (Arimidex®), retinoids suchas N-[4-hydroxyphenyl]retinamide (4-HPR, fenretinide), nonsteriodalantiinflammatory agents (NSAIDs) such as aspirin and sulindac, celecoxib(COX-2 inhibitor), defluoromethylornithing (DFMO), ursodeoxycholic acid,3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, EKI-785(EGFR inhibitor), bevacizumab (antibody to VEGF-receptor), cetuximab(antibody to EGFR), retinol such as vitamin A, beta-carotene, 13-cisretinoic acid, isotretinoin and retinyl palmitate, α-tocopherol,interferon, oncolytic adenovirus dl1520 (ONYX-015), gefitinib,etretinate, finasteride, indole-3-carbinol, resveratrol, chlorogenicacid, raloxifene, and oltipraz.

Compositions for Facilitating Entry of Compounds into Cancer Cells andTumors

The present invention relates to methods and materials for delivering acargo compound into a cell. Delivery of the cargo compound according tothis invention is accomplished by the use of a suitable transportpolypeptide. In one embodiment of the invention, the cargo compound islinked to the transport polypeptide. Suitable transport peptides includea cupredoxin, or a fragment of a cupredoxin containing a “cupredoxinentry domain”. The term “cupredoxin entry domain” refers to a fragmentof a cupredoxin that includes the amino sequence that is required forthe entry of cupredoxin into a mammalian cancer cell. Cargo compoundsdelivered by the present invention include, but are not limited to,proteins, lipoproteins, polypeptides, peptides, polysaccharides, nucleicacids, including RNA, DNA and anti-sense nucleic acids, dyes,fluorescent and radioactive tags, microparticles or nanoparticles,toxins, inorganic and organic molecules, small molecules, and drugs (forexample, chemopreventive drugs). In some embodiments, the drugs andtoxins kill tumor cells.

In one embodiment of the invention, the cupredoxin is an azurin, such asazurin from Pseudomonas aeruginosa (SEQ ID NO: 1). In other embodimentsof the invention, the cupredoxin is a plastocyanin, a rusticyanin, or apseudoazurin, among others. In specific embodiments, the azurin is fromPseudomonas aeruginosa, Pseudomonas syringa, Neisseria meningitides,Neisseria gonorrhoeae, Vibrio parahaemolyticus or Bordetellabronchiseptica, among others.

In one embodiment, a cargo compound is delivered to kill or retard cellcycle progression in a cell, such as a cancer cell. Such a cancer cellcan be, for example, an osteosarcoma cell, lung carcinoma cell, coloncarcinoma cell, lymphoma cell, leukemia cell, soft tissue sarcoma cellor breast, liver, bladder or prostate carcinoma cell, among others. Forexample, the cargo compound can be a cell cycle control protein, such asp53; a cyclin-dependent kinase inhibitor, such as p16, p21 or p27; asuicide protein such as thymidine kinase or nitroreductase; a cytokineor other immunomodulatory protein such as interleukin 1, interleukin 2or granulocyte-macrophage colony stimulating factor (GM-CSF); or atoxin, such as Pseudomonas aeruginosa exotoxin A, among others. In otherembodiments, a biologically active fragment of one of the above classesof compounds is delivered. In another embodiment, the cargo compound isdelivered in order to generate an image of the target tissue. Forexample, the target tissue can be a cancer and the cargo compound can beone commonly used to generate an image for detection by X-ray computedtomography (CT), Magnetic Resonance Imaging (MRI) and ultrasound. Inthese embodiments, the cargo compound is a gamma ray or positronemitting radioisotope, a magnetic resonance imaging contrast agent, anX-ray contrast agent, or an ultrasound contrast agent.

The invention further includes methods of selectively introducing DNA orRNA into a mammalian cancer cell. In such embodiments, the DNA or RNA isthe cargo compound. In some embodiments, the method includes providingp18 or p28 coupled to DNA or RNA and introducing the compound into thebody of a mammal. In some embodiments, the DNA or RNA is a gene or afragment of a gene. In some embodiments, the DNA or RNA has atherapeutic effect once introduced into a mammalian cell.

Cupredoxin Entry Domain

The invention provides for a protein transduction domain that allows forthe preferential entry of peptides into cancer cells, as well astransport of linked cargo into mammalian cancer cells but notnon-cancerous cells. It has been discovered that cupredoxin proteinscomprise a protein transduction domain, the cupredoxin entry domain,which facilitates the entry of linked cargo into mammalian cancer cells.In some embodiments, the entire cupredoxin protein can be used tofacilitate the transport linked cargo selectively into cancer cells. Inother embodiments, a portion of a cupredoxin can be used to transportlinked cargo into cancer cells. In some embodiments, the cupredoxinentry domain consists of a region of a cupredoxin that is less that thefull length wild-type protein. In some embodiments, the cupredoxin entrydomain consists of more than about 10 residues, about 15 residues orabout 20 residues of a cupredoxin. In some embodiments, the cupredoxinentry domain consists of not more than about 50 residues, about 40residues or about 30 residues of a cupredoxin. In some embodiments, thecupredoxin entry domain has at least about 90% amino acid sequenceidentity, at least about 95% amino acid sequence identity or at leastabout 99% amino acid sequence identity to a cupredoxin.

In some embodiments, the cupredoxin entry domain is an azurin entrydomain. In one embodiment of the present invention, azurin entry domaincontains at least amino acids 50 to 77 of Pseudomonas aeruginosa azurin,p28 (SEQ ID NO: 2). In another embodiments of the invention, thecupredoxin entry domain contains at least amino acids 36 to 77 ofPseudomonas aeruginosa azurin (SEQ ID NO: 27). In another embodiment ofthe invention, the cupredoxin entry domain contains at least amino acids36 to 89 of Pseudomonas aeruginosa azurin (SEQ ID NO: 28). In anotherembodiment of the invention, the cupredoxin entry domain contains atleast amino acids 36 to 128 of Pseudomonas aeruginosa azurin (SEQ ID NO:29). In yet another embodiment of the invention, the cupredoxin entrydomain contains at least amino acids 50 to 67 of Pseudomonas aeruginosaazurin, p18 (SEQ ID NO: 25). In another embodiment of the invention, thecupredoxin entry domain contains at least amino acids 53 to 70 ofPseudomonas aeruginosa azurin (SEQ ID NO: 30). In yet another embodimentof the invention, the cupredoxin entry domain contains at least aminoacids 53 to 64 of Pseudomonas aeruginosa azurin (SEQ ID NO: 31).

The Examples described herein, particularly Example 19, demonstrate thatthe C-terminal region of p28, not present on p18 (amino acids 50-67) ismost likely to contact specific residues on the cell membrane andprovide access to the cell. As such, in another embodiment of theinvention, the cupredoxin entry domain is an azurin entry domaincontaining at least amino acids 66-77 of p28 (SEQ ID NO. 35). In anotherembodiment of the invention, the cupredoxin entry domain is an azurinentry domain containing at least amino acids 68-77 of p28 (SEQ ID NO.36). In another embodiment of the invention, the cupredoxin entry domainis an azurin entry domain containing at least amino acids 67-77 of p28(SEQ ID NO. 37). In another embodiment of the invention, the cupredoxinentry domain comprises one or more of the amino acids located atpositions 69, 70, 75, 76, and 85 of SEQ ID NO. 2. In another embodiment,the cupredoxin entry domain comprises amino acids 69, 70, 75, 76, and 85of SEQ ID NO. 2.

In another embodiment of the invention, the cupredoxin entry domain isan entry domain from a cupredoxin other than P. aeruginosa azurin. Indifferent embodiments, the cupredoxin entry domain may be a fragment ofplastocyanin from the cyanobacterium Phormidium laminosum (SEQ ID NO:3), rusticyanin from Thiobacillus ferrooxidans (SEQ ID NO: 4);pseudoazurin from Achromobacter cycloclastes (SEQ ID NO: 5), azurin fromPseudomonas syringae (SEQ ID NO: 21), azurin from Neisseria meningitidis(SEQ ID NO: 10), azurin from Vibrio parahaemolyticus (SEQ ID NO: 8), oran auracyanin from Chloroflexus aurantiacus (SEQ ID NO: 15 and 16).

In another embodiment of the invention, the cupredoxin entry domaincontains at least amino acids 57 to 89 of auracyanin B of Chloroflexusaurantiacus (SEQ ID NO: 20). In another embodiment of the invention, thecupredoxin entry domain contains at least amino acids 51-77 ofPseudomonas syringae azurin (SEQ ID NO: 21). In another embodiment ofthe invention, the cupredoxin entry domain contains at least amino acids89-115 of Neisseria meningitidis Laz (SEQ ID NO: 22). In anotherembodiment of the invention, the cupredoxin entry domain contains atleast amino acids 52-78 of Vibrio parahaemolyticus azurin (SEQ ID NO:23). In another embodiment of the invention, the cupredoxin entry domaincontains at least amino acids 51-77 of Bordetella bronchiseptica azurin(SEQ ID NO: 24).

Modification of a Cupredoxin Entry Domain

In another embodiment of the present invention, a cupredoxin entrydomain is chemically modified or genetically altered to produce variantsthat retain the ability to preferentially enter and/or transport a cargocompound into a cell. For example, Example 14 shows that Pseudomonasaeruginosa azurin having proline residues introduced at positions 54, 61and 70 retains its ability to enter UISO-Mel-2 cells.

In another embodiment, the cupredoxin entry domain comprises a conservedamino acid sequence DGXXXXXDXXYXKXXD (SEQ ID NO: 32) or DGXXXXDXXYXKXXD(SEQ ID NO: 33) where D is aspartic acid, G is glycine, Y is tyrosine, Kis lysine and X is any amino acid. See Example 17.

Variants of a cupredoxin entry domain may be synthesized by standardtechniques. Derivatives are amino acid sequences formed from nativecompounds either directly or by modification or partial substitution.Analogs are amino acid sequences that have a structure similar, but notidentical, to the native compound but differ from it in respect tocertain components or side chains. Analogs may be synthesized or from adifferent evolutionary origin.

Variants may be full length or other than full length, if the derivativeor analog contains a modified amino acid. Variants of a cupredoxin entrydomain include, but are not limited to, molecules comprising regionsthat are substantially homologous to the cupredoxin entry domain by atleast about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% identity over anamino acid sequence of identical size or when compared to an alignedsequence in which the alignment is performed by a homology algorithm.

In another embodiment, the variants of a cupredoxin entry domain have asignificant structural similarity to P. aeruginosa azurin residues50-77, p28 (SEQ ID NO: 2). In other embodiments, the variants of acupredoxin entry domain have a significant structural similarity to P.aeruginosa azurin residues 50-67, p18 (SEQ ID NO: 25). Examples ofstudies that determine significant structural homology betweencupredoxins and other proteins include Toth et al. (Developmental Cell1:82-92 (2001)). Specifically, significant structural homology between avariant of the cupredoxin entry domain and P. aeruginosa azurin residues50-77 (SEQ ID NO: 2) is determined by using the VAST algorithm (Gibratet al., Curr Opin Struct Biol 6:377-385 (1996); Madej et al., Proteins23:356-3690 (1995)). In specific embodiments, the VAST p value from astructural comparison of a variant of the cupredoxin entry domain and P.aeruginosa azurin residues 50-77 (SEQ ID NO: 2) is less than about 10⁻³,less than about 10⁻⁵, or less than about 10⁻⁷. In other embodiments,significant structural homology between a variant of the cupredoxinentry domain and P. aeruginosa azurin residues 50-77 (SEQ ID NO: 2) canbe determined by using the DALI algorithm (Holm & Sander, J. Mol. Biol.233:123-138 (1993)). In specific embodiments, the DALI Z score for apairwise structural comparison is at least about 3.5, at least about7.0, or at least about 10.0.

Modifications to the cupredoxin entry domain can be made using methodsknown in the art such as oligonucleotide-mediated (site-directed)mutagenesis, alanine scanning, PCR mutagenesis, and the methods andtechniques disclosed herein. Site-directed mutagenesis (Carter, BiochemJ. 237:1-7 (1986); Zoller and Smith, Methods Enzymol. 154:329-50(1987)), cassette mutagenesis, restriction selection mutagenesis (Wellset al., Gene 34:315-23 (1985)) or other known techniques can beperformed on the cloned DNA to produce a cupredoxin entry domain variantnucleic acid. In addition, nucleotides encoding entry domains withstructural similarity to that of the cupredoxin entry domains may besynthesized by methods that are well known in the art. Further, proteinmolecules that are wild type or variant cupredoxin entry domains may besynthesized by methods that are well known in the art.

Nucleic Acids Coding for a Cupredoxin Entry Domain and Complex of aCupredoxin Entry Domain Linked to a Cargo Compound

In another aspect, the present invention provides a nucleic acidmolecule encoding a fusion protein comprising a cupredoxin entry domainlinked to a cargo compound, where the cargo compound is a protein orpeptide. The nucleic acid molecule according to the invention can beprepared by a combination of known techniques in the art. For instance,nucleic acid sequences for the cupredoxin entry domain and the cargocompound can individually be prepared by chemical synthesis or cloning.The nucleic acid sequences are then ligated in order with a ligase togive a nucleic acid molecule of interest.

Methods of Delivering a Cargo Compound Using a Cupredoxin Entry Domain

Many arginine-rich peptides are known to translocate through mammaliancell membranes and carry protein cargo compounds inside such cells.Suzuki, T., et al. J. Biol. Chem. 277:2437-43 (2002). For example, ashort arginine-rich 11 amino acid (amino acids 47-57) segment of HIV Tatprotein allows transport of cargo proteins into mammalian cells.Schwarze, S R., et al. Trends Cell Biol. 10:290-95 (2000). Syntheticentry domains that strengthen the alpha-helical content and optimize theplacement of arginine residues have been shown to have enhancedpotential as protein transduction domains. Ho, A., et al. Cancer Res.61:474-77 (2001). In comparison, P. aeruginosa azurin has a singlearginine residue.

In some embodiments, the present invention encompasses the use of thosecupredoxin fragments that facilitate the entry of a cargo compound intoa cell, such as p18 (SEQ ID NO. 25) and p28 (SEQ ID NO. 2). Suchfragments may be determined by any method that identifies thosefragments required for entry into a cell. In one such method, acupredoxin fragment is linked to a marker substance and a test performedto determine whether the cupredoxin fragment enters a cell. Such methodsmay be used to identify suitable fragments of the cupredoxins discussedabove.

In various embodiments of the present invention, the cargo compound isattached to a cupredoxin or a fragment thereof, such as azurin from P.aeruginosa (SEQ ID NO: 1); plastocyanin from the cyanobacteriumPhormidium laminosum (SEQ ID NO: 3); rusticyanin from Thiobacillusferrooxidans (SEQ ID NO: 4); or pseudoazurin from Achromobactercycloclastes (SEQ ID NO: 5), a fragment of an azurin from Pseudomonassyringae (SEQ ID NO: 21), azurin from Neisseria meningitidis (SEQ ID NO:10), azurin from Vibrio parahaemolyticus (SEQ ID NO: 19), azurin fromBordelella bronchiseptica (SEQ ID NO: 8), auracyanin A and B fromChloroflexus aurantiacus (SEQ ID NO. 15 and 16), among other azurin andazurin-like proteins. In other embodiments, the cargo is linked to acupredoxin entry domain such as p28 (SEQ ID NO: 2), p18 (SEQ ID NO: 25),or any one of SEQ ID NOs: 35-37.

In various embodiments of the present invention, a cupredoxin entrydomain delivers a cargo compound into a cell in vitro, ex vivo or invivo. For example, delivery may be achieved in vitro by adding a complexof a cupredoxin entry domain and a cargo compound to a cell culture,such as a pap smear. Alternatively, delivery may be achieved ex vivo byadding the complex to a sample removed from a patient, for example,blood, tissue, or bone marrow, and returning the treated sample to thepatient. Delivery may also be achieved by administration of the complexdirectly to a patient. The methods of the present invention may be usedfor therapeutic, prophylactic, diagnostic or research purposes. Cargocompounds delivered by the present invention include, but are notlimited to, proteins, lipoproteins, polypeptides, peptides,polysaccharides, nucleic acids, including anti-sense nucleic acids,dyes, microparticles or nanoparticles, toxins, organic and inorganicmolecules, small molecules, and drugs.

In one embodiment, a detectable substance, for example, a fluorescentsubstance, such as green fluorescent protein; a luminescent substance;an enzyme, such as β-galactosidase; or a radiolabelled or biotinylatedprotein is delivered to confer a detectable phenotype to a cell.Similarly, microparticles or nanoparticles labeled with a detectablesubstance, for example, a fluorescent substance, can be delivered. Oneexample of suitable nanoparticles is found in U.S. Pat. No. 6,383,500,issued May 7, 2002, which is hereby expressly incorporated by reference.Many such detectable substances are known to those skilled in the art.

In some embodiments, the cargo compound is a detectable substance thatis suitable for X-ray computed tomography, magnetic resonance imaging,ultrasound imaging or radionuclide scintigraphy. In these embodiments,the cargo compound is administered to the patient for purposes ofdiagnosis. A contrast agent is administered as a cargo compound toenhance the image obtained by X-ray CT, MRI and ultrasound. Theadministration of a radionuclide cargo compound that is targeted totumor tissue via the cupredoxin entry domain can be used forradionuclide scinitigraphy. In some embodiments, the cupredoxin entrydomain may contain the radionucleotide with or without a cargo compound.In other embodiments, the cargo compound is a gamma ray or positronemitting radioisotope, a magnetic resonance imaging contract agent, anX-ray contrast agent, or an ultrasound contrast agent.

Ultrasound contrast agents suitable for use as cargo compounds include,but are not limited to, a microbubble of a biocompatible gas, a liquidcarrier, and a surfactant microsphere, further comprising an optionallinking moiety, L_(n), between the targeting moieties and themicrobubble. In this context, the term liquid carrier means aqueoussolution and the term surfactant means any amphiphilic material whichproduces a reduction in interfacial tension in a solution. A list ofsuitable surfactants for forming surfactant microspheres is disclosed inEP0727225A2, herein expressly incorporated by reference. The termsurfactant microsphere includes nanospheres, liposomes, vesicles and thelike. The biocompatible gas can be air, or a fluorocarbon, such as aC₃-C₅ perfluoroalkane, which provides the difference in echogenicity andthus the contrast in ultrasound imaging. The gas is encapsulated orcontained in the microsphere to which is attached the cupredoxin entrydomain, optionally via a linking group. The attachment can be covalent,ionic or by van der Waals forces. Specific examples of such contrastagents include lipid encapsulated perfluorocarbons with a plurality oftumor neovasculature receptor binding peptides, polypeptides orpeptidomimetics.

X-ray contrast agents suitable for use as cargo compounds include, butare not limited to, one or more X-ray absorbing or “heavy” atoms ofatomic number 20 or greater, further comprising an optional linkingmoiety, L_(n), between the cupredoxin entry domain and the X-rayabsorbing atoms. The frequently used heavy atom in X-ray contrast agentsis iodine. Recently, X-ray contrast agents comprised of metal chelates(e.g., U.S. Pat. No. 5,417,959) and polychelates comprised of aplurality of metal ions (e.g., U.S. Pat. No. 5,679,810) have beendisclosed. More recently, multinuclear cluster complexes have beendisclosed as X-ray contrast agents (e.g., U.S. Pat. No. 5,804,161, PCTWO91/14460, and PCT WO 92/17215).

MRI contrast agents suitable for use as cargo compounds include, but arenot limited to, one or more paramagnetic metal ions, further comprisingan optional linking moiety, L_(n), between the cupredoxin entry domainand the paramagnetic metal ions. The paramagnetic metal ions are presentin the form of metal complexes or metal oxide particles. U.S. Pat. Nos.5,412,148, and 5,760,191, describe examples of chelators forparamagnetic metal ions for use in MRI contrast agents. U.S. Pat. No.5,801,228, U.S. Pat. No. 5,567,411, and U.S. Pat. No. 5,281,704,describe examples of polychelants useful for complexing more than oneparamagnetic metal ion for use in MRI contrast agents. U.S. Pat. No.5,520,904, describes particulate compositions comprised of paramagneticmetal ions for use as MRI contrast agents.

In another embodiment, a cargo compound is delivered to kill or retardcell cycle progression in a cell, such as a cancer cell. Such a cancercell can be, for example, an osteosarcoma cell, lung carcinoma cell,colon carcinoma cell, lymphoma cell, leukemia cell, soft tissue sarcomacell or breast, liver, bladder or prostate carcinoma cell. For example,the cargo compound can be a cell cycle control protein, such as p53; acyclin-dependent kinase inhibitor, such as p16, p21 or p27; a suicideprotein such as thymidine kinase or nitroreductase; a cytokine or otherimmunomodulatory protein such as interleukin 1, interleukin 2 orgranulocyte-macrophage colony stimulating factor (GM-CSF); or a toxin,such as Pseudomonas aeruginosa exotoxin A. In other embodiments, abiologically active fragment of one of the above classes of compounds isdelivered.

In yet another embodiment, the cargo compound is a nucleic acid. In someembodiments the nucleic acid codes for one of the above classes ofcompounds. In yet another embodiment, the cargo compound is a drug usedto treat cancer. Such drugs include, for example, 5-fluorouracil;Interferon α; Methotrexate; Tamoxifen; and Vincrinstine. The aboveexamples are provided for illustration only, many other such compoundsare known to those skilled in the art. In other embodiments, the nucleicacid is useful for gene therapy.

Cargo compounds suitable for treating cancer include, but not limitedto, alkylating agents such as nitrogen mustards, alkyl sulfonates,nitrosoureas, ethylenimines, and triazenes; antimetabolites such asfolate antagonists, purine analogues, and pyrimidine analogues;antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin,and plicamycin; enzymes such as L-asparaginase; farnesyl-proteintransferase inhibitors; 5.alpha.-reductase inhibitors; inhibitors of17.beta.-hydroxysteroid dehydrogenase type 3; hormonal agents such asglucocorticoids, estrogens/antiestrogens, androgens/antiandrogens,progestins, and luteinizing hormone-releasing hormone antagonists,octreotide acetate; microtubule-disruptor agents, such as ecteinascidinsor their analogs and derivatives; microtubule-stabilizing agents such astaxanes, for example, paclitaxel (Taxol™), docetaxel (Taxotere™), andtheir analogs, and epothilones, such as epothilones A-F and theiranalogs; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, taxanes; and topiosomerase inhibitors;prenyl-protein transferase inhibitors; and miscellaneous agents such ashydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinumcoordination complexes such as cisplatin and carboplatin; and otheragents used as anti-cancer and cytotoxic agents such as biologicalresponse modifiers, growth factors; immune modulators and monoclonalantibodies.

Representative examples of these classes of anti-cancer and cytotoxicagents include but are not limited to mechlorethamine hydrochloride,cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan,carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine,methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin,cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride,daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D,safracins, saframycins, quinocarcins, discodermolides, vincristine,vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate,teniposide, paclitaxel, tamoxifen, estramustine, estramustine phosphatesodium, flutamide, buserelin, leuprolide, pteridines, diyneses,levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim,sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride,betamethosone, gemcitabine hydrochloride, altretamine, and topoteca andany analogs or derivatives thereof.

Preferred members of these classes include, but are not limited to,paclitaxel, cisplatin, carboplatin, doxorubicin, caminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or pofiromycin, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

Examples of anticancer and other cytotoxic agents useful as cargocompounds include the following: epothilone derivatives as found inGerman Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO98/38192, WO 99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO99/07692, WO 99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO99/54318, WO 99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO00/00485; cyclin dependent kinase inhibitors as found in WO 99/24416(see also U.S. Pat. No. 6,040,321); and prenyl-protein transferaseinhibitors as found in WO 97/30992 and WO 98/54966; and agents such asthose described generically and specifically in U.S. Pat. No. 6,011,029(the compounds of which U.S. patent can be employed together with anyNHR modulators (including, but not limited to, those of presentinvention) such as AR modulators, ER modulators, with LHRH modulators,or with surgical castration, especially in the treatment of cancer).

The above other therapeutic agents, when employed as cargo compoundswith the compounds of the present invention, may be used, for example,in those amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art.

Pharmaceutical Compositions Containing a Cupredoxin Entry Domain

Pharmaceutical compositions containing, comprising, or consisting of acupredoxin entry domain, as well as pharmaceutical compositionscontaining complex of a cupredoxin entry domain linked to a cargocompound, can be manufactured in any conventional manner, e.g., byconventional mixing, dissolving, granulating, dragee-making,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thecomplex can be readily combined with a pharmaceutically acceptablecarrier well-known in the art. Such carriers enable the preparation tobe formulated as a tablet, pill, dragee, capsule, liquid, gel, syrup,slurry, suspension, and the like. Suitable excipients can also include,for example, fillers and cellulose preparations. Other excipients caninclude, for example, flavoring agents, coloring agents, detackifiers,thickeners, and other acceptable additives, adjuvants, or binders.

Such compositions can be used in, for example, the detection or imagingof a cell type or in the treatment of a condition related to cell deathor in the prevention thereof. The compositions can be administered in anamount sufficient to prevent or treat a condition related to resistanceto cell death. As used herein, the term “a condition related toresistance to cell death” refers to a disease, state, or ailmentcharacterized by at least a tendency for prolonged cell life whencompared with a healthy cell of like kind as determined by a reasonable,skilled physician or clinician. Typically, the host organism is amammal, such as a human or animal.

Administration of Compositions Containing a Cupredoxin Entry Domain

Compositions containing a cupredoxin entry domain can be administered byany suitable route, for example, by oral, buccal, inhalation,sublingual, rectal, vaginal, transurethral, nasal, topical,percutaneous, i.e., transdermal or parenteral (including intravenous,intramuscular, subcutaneous and intracoronary administration). Thecompositions and pharmaceutical formulations thereof can be administeredin any amount effective to achieve its intended purpose. Whenadministrated to treat a condition related to resistance to cell death,the composition is administered in a therapeutically effective amount. A“therapeutically effective amount” is an amount effective to preventdevelopment of, or to alleviate the existing symptoms of, the subjectbeing treated. Determination of a therapeutically effective amount iswell within the capability of those skilled in the art.

The appropriate dosage will, of course, vary depending upon, forexample, the compound containing the cupredoxin entry domain employed,the host, the mode of administration and the nature and severity of theconditions being treated or diagnosed. However, in one embodiment of themethods of the present invention, satisfactory treatment results inhumans are indicated to be obtained at daily dosages from about 0.001 toabout 20 mg/kg of body weight of the compound containing the cupredoxinentry domain. In one embodiment, an indicated daily dosage for treatmentin humans may be in the range from about 0.7 mg to about 1400 mg of acompound containing the cupredoxin entry domain convenientlyadministered, for example, in daily doses, weekly doses, monthly doses,and/or continuous dosing. Daily doses can be in discrete dosages from 1to 12 times per day. Alternatively, doses can be administered everyother day, every third day, every fourth day, every fifth day, everysixth day, every week, and similarly in day increments up to 31 days.Dosing can be continuous, intermittent or a single dose, using anyapplicable dosing form, including tablet, patches, i.v. administrationand the like. More specifically, the composition is administered in atherapeutically effective amount. In specific embodiments, thetherapeutically effective amount is from about 0.01-20 mg/kg of bodyweight. In specific embodiments, the dose level is about 10 mg/kg/day,about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30mg/kg/day, about 35 mg/kg/day, about 40 mg/kg/day, about 45 mg/kg/day orabout 50 mg/kg/day.

The method of introducing compounds containing the cupredoxin entrydomain to patients is, in some embodiments, co-administration with otherdrugs known to treat cancer. Such methods are well-known in the art. Ina specific embodiment, the compounds containing the cupredoxin entrydomain are part of an cocktail or co-dosing containing or with otherdrugs for treating cancer. Such drugs include, for example, those listedherein and specifically 5-fluorouracil; Interferon α; Methotrexate;Tamoxifen; and Vincrinstine. The above examples are provided forillustration only, many other such compounds are known to those skilledin the art.

Nucleic acid molecules encoding a cupredoxin entry domain or a fusionprotein combining a either entry domain and a cargo compound can beinserted into vectors and used as gene therapy vectors. Gene therapyvectors can be delivered to a subject by, for example, intravenousinjection, local administration (Nabel et al., U.S. Pat. No. 5,328,4701994. USA), or by stereotactic injection (Chen et al., Proc Natl AcadSci USA, vol. 91, pp 3054-57 (1994)). The pharmaceutical preparation ofa gene therapy vector can include an acceptable diluent or can comprisea slow release matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

In one aspect, the composition is delivered as DNA such that the complexis generated in situ. In one embodiment, the DNA is “naked,” asdescribed, for example, in Ulmer et al., Science 259:1745-49 (1993) andreviewed by Cohen, Science 259 1691-92 (1993). The uptake of naked DNAmay be increased by coating the DNA onto a carrier, e.g. a biodegradablebead, which is efficiently transported into the cells. In such methods,the DNA may be present within any of a variety of delivery systems knownto those of ordinary skill in the art, including nucleic acid expressionsystems, bacterial and viral expression systems. Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. See, e.g., WO90/11092, WO93/24640, WO93/17706, and U.S. Pat. No. 5,736,524.

Vectors, used to shuttle genetic material from organism to organism, canbe divided into two general classes: Cloning vectors are replicatingplasmid or phage with regions that are non-essential for propagation inan appropriate host cell and into which foreign DNA can be inserted; theforeign DNA is replicated and propagated as if it were a component ofthe vector. An expression vector (such as a plasmid, yeast, or animalvirus genome) is used to introduce foreign genetic material into a hostcell or tissue in order to transcribe and translate the foreign DNA,such as the DNA of the composition. In expression vectors, theintroduced DNA is operably-linked to elements such as promoters thatsignal to the host cell to transcribe the inserted DNA. Some promotersare exceptionally useful, such as inducible promoters that control genetranscription in response to specific factors. Operably-linking acomposition polynucleotide to an inducible promoter can control theexpression of the wt-azurin entry domain composition polypeptide orfragments. Examples of classic inducible promoters include those thatare responsive to α-interferon, heat shock, heavy metal ions, andsteroids such as glucocorticoids (Kaufman, Methods Enzymol. 185:487-511(1990)) and tetracycline. Other desirable inducible promoters includethose that are not endogenous to the cells in which the construct isbeing introduced, but, however, are responsive in those cells when theinduction agent is exogenously supplied. In general, useful expressionvectors are often plasmids. However, other forms of expression vectors,such as viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses) are contemplated.

Vector choice is dictated by the organism or cells being used and thedesired fate of the vector. In general, vectors comprise signalsequences, origins of replication, marker genes, enhancer elements,promoters, and transcription termination sequences.

Kits Comprising a Cupredoxin Entry Domain-Cargo Compound Complex

In another aspect, the invention provides kits containing one or more ofthe following in a package or container: (1) a reagent comprising acupredoxin entry domain on its own, such as p18 or p28, or linked to acargo compound; (2) a reagent containing a pharmaceutically acceptableadjuvant or excipient; (3) a vehicle for administration, such as asyringe; (4) instructions for administration. Embodiments in which twoor more of components (1)-(4) are found in the same container are alsocontemplated.

Pharmaceutical Compositions Comprising Cupredoxin, a Cupredoxin EntryDomain, a Cupredoxin Entry Domain—Cargo Compound Complex, or Variant,Derivative or Structural Equivalent Thereof

Pharmaceutical compositions comprising cupredoxin or variant, derivativeor structural equivalents thereof, can be manufactured in anyconventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, emulsifying, encapsulating, entrapping, orlyophilizing processes. The substantially pure or pharmaceutical gradecupredoxin or variants, derivatives and structural equivalents thereofcan be readily combined with a pharmaceutically acceptable carrierwell-known in the art. Such carriers enable the preparation to beformulated as a tablet, pill, dragee, capsule, liquid, gel, syrup,slurry, suspension, and the like. Suitable carriers or excipients canalso include, for example, fillers and cellulose preparations. Otherexcipients can include, for example, flavoring agents, coloring agents,detackifiers, thickeners, and other acceptable additives, adjuvants, orbinders. In some embodiments, the pharmaceutical preparation issubstantially free of preservatives. In other embodiments, thepharmaceutical preparation may contain at least one preservative.General methodology on pharmaceutical dosage forms is found in Ansel etal., Pharmaceutical Dosage Forms and Drug Delivery Systems (LippencottWilliams & Wilkins, Baltimore Md. (1999)).

The composition comprising a cupredoxin or variant, derivative orstructural equivalent thereof used in the invention may be administeredin a variety of ways, including by injection (e.g., intradermal,subcutaneous, intramuscular, intraperitoneal and the like), byinhalation, by topical administration, by suppository, by using atransdermal patch or by mouth. General information on drug deliverysystems can be found in Ansel et al., id. In some embodiments, thecomposition comprising a cupredoxin or variant, derivative or structuralequivalent thereof can be formulated and used directly as injectibles,for subcutaneous and intravenous injection, among others. The injectableformulation, in particular, can advantageously be used to treat patientsthat are appropriate for chemopreventive therapy. The compositioncomprising a cupredoxin or variant, derivative or structural equivalentthereof can also be taken orally after mixing with protective agentssuch as polypropylene glycols or similar coating agents.

When administration is by injection, the cupredoxin or variant,derivative or structural equivalent thereof may be formulated in aqueoussolutions, specifically in physiologically compatible buffers such asHanks solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the cupredoxin or variant,derivative or structural equivalent thereof may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. In some embodiments, the pharmaceutical composition does notcomprise an adjuvant or any other substance added to enhance the immuneresponse stimulated by the peptide. In some embodiments, thepharmaceutical composition comprises a substance that inhibits an immuneresponse to the peptide.

When administration is by intravenous fluids, the intravenous fluids foruse administering the cupredoxin or variant, derivative or structuralequivalent thereof may be composed of crystalloids or colloids.Crystalloids as used herein are aqueous solutions of mineral salts orother water-soluble molecules. Colloids as used herein contain largerinsoluble molecules, such as gelatin. Intravenous fluids may be sterile.

Crystalloid fluids that may be used for intravenous administrationinclude but are not limited to, normal saline (a solution of sodiumchloride at 0.9% concentration), Ringer's lactate or Ringer's solution,and a solution of 5% dextrose in water sometimes called D5W, asdescribed in Table 2.

TABLE 2 Composition of Common Crystalloid Solutions Solution Other Name[Na⁺] [Cl⁻] [Glucose] D5W 5% Dextrose 0 0 252 ⅔ & ⅓ 3.3% Dextrose/ 51 51168 0.3% saline Half-normal 0.45% NaCl 77 77 0 saline Normal saline 0.9%NaCl 154 154 0 Ringer's Ringer's 130 109 0 lactate* solution *Ringer'slactate also has 28 mmol/L lactate, 4 mmol/L K⁺ and 3 mmol/L Ca²⁺.

When administration is by inhalation, the cupredoxin or variant,derivative or structural equivalent thereof may be delivered in the formof an aerosol spray from pressurized packs or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator may beformulated containing a powder mix of the proteins and a suitable powderbase such as lactose or starch.

When administration is by topical administration, the cupredoxin orvariant, derivative or structural equivalent thereof may be formulatedas solutions, gels, ointments, creams, jellies, suspensions, and thelike, as are well known in the art. In some embodiments, administrationis by means of a transdermal patch. When administration is bysuppository (e.g., rectal or vaginal), cupredoxin or variants andderivatives thereof compositions may also be formulated in compositionscontaining conventional suppository bases.

When administration is oral, a cupredoxin or variant, derivative orstructural equivalent thereof can be readily formulated by combining thecupredoxin or variant, derivative or structural equivalent thereof withpharmaceutically acceptable carriers well known in the art. A solidcarrier, such as mannitol, lactose, magnesium stearate, and the like maybe employed; such carriers enable the cupredoxin and variants,derivatives or structural equivalent thereof to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, cellulosepreparation, granulating agents, and binding agents.

Other convenient carriers, as well-known in the art, also includemultivalent carriers, such as bacterial capsular polysaccharide, adextran or a genetically engineered vector. In addition,sustained-release formulations that include a cupredoxin or variant,derivative or structural equivalent thereof allow for the release ofcupredoxin or variant, derivative or structural equivalent thereof overextended periods of time, such that without the sustained releaseformulation, the cupredoxin or variant, derivative or structuralequivalent thereof would be cleared from a subject's system, and/ordegraded by, for example, proteases and simple hydrolysis beforeeliciting or enhancing a therapeutic effect.

The half-life in the bloodstream of the peptides of the invention can beextended or optimized by several methods well known to those in the art.The peptide variants of the invention may include, but are not limitedto, various variants that may increase their stability, specificactivity, longevity in the bloodstream, and/or decrease immunogenicityof the cupredoxin, while retaining the ability of the peptide to inhibitthe development of premalignant lesions in mammalian cells, tissues andanimals. Such variants include, but are not limited to, those whichdecrease the hydrolysis of the peptide, decrease the deamidation of thepeptide, decrease the oxidation, decrease the immunogenicity, increasethe structural stability of the peptide or increase the size of thepeptide. Such peptides also include circularized peptides (see Monk etal., BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res.63(5):409-19 (2004)), D,L-peptides (diastereomer), Futaki et al., J.Biol. Chem. February 23; 276(8):5836-40 (2001); Papo et al., Cancer Res.64(16):5779-86 (2004); Miller et al., Biochem. Pharmacol. 36(1):169-76,(1987)); peptides containing unusual amino acids (see Lee et al., J.Pept. Res. 63(2):69-84 (2004)), N- and C-terminal modifications (seeLabrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), hydrocarbonstapling (see Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892(2000); Walenski et al., Science 305:1466-1470 (2004)) and PEGylation.

In various embodiments, the pharmaceutical composition includes carriersand excipients (including but not limited to buffers, carbohydrates,mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents, suspending agents,thickening agents and/or preservatives), water, oils, saline solutions,aqueous dextrose and glycerol solutions, other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like. It will be recognized that, while any suitablecarrier known to those of ordinary skill in the art may be employed toadminister the compositions of this invention, the type of carrier willvary depending on the mode of administration. Compounds may also beencapsulated within liposomes using well-known technology. Biodegradablemicrospheres may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.

The pharmaceutical compositions may be sterilized by conventional,well-known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration.

Administration of Cupredoxin or Variant, Derivative or StructuralEquivalent Thereof

The cupredoxin or variant, derivative or structural equivalent thereof,such as p18 or p28, can be administered formulated as pharmaceuticalcompositions and administered by any suitable route, for example, byoral, buccal, inhalation, sublingual, rectal, vaginal, transurethral,nasal, topical, percutaneous, i.e., transdermal or parenteral (includingintravenous, intramuscular, subcutaneous and intracoronary) or vitreousadministration. The pharmaceutical formulations thereof can beadministered in any amount effective to achieve its intended purpose.More specifically, the composition is administered in a therapeuticallyeffective amount. In specific embodiments, the therapeutically effectiveamount is generally from about 0.01-20 mg/day/kg of body weight.

The compounds comprising cupredoxin or variant, derivative or structuralequivalent thereof are useful for the prevention of cancer, alone or incombination with other active agents and/or cargo compounds. Theappropriate dosage will, of course, vary depending upon, for example,the compound of cupredoxin or variant, derivative or structuralequivalent thereof employed, the host, the mode of administration andthe nature and severity of the potential cancer. However, in general,satisfactory results in humans are indicated to be obtained at dailydosages from about 0.01-20 mg/kg of body weight. An indicated dailydosage in humans is in the range from about 0.7 mg to about 1400 mg of acompound of cupredoxin or variant, derivative or structural equivalentthereof conveniently administered, for example, in daily doses, weeklydoses, monthly doses, and/or continuous dosing. Daily doses can be indiscrete dosages from 1 to 12 times per day. Alternatively, doses can beadministered every other day, every third day, every fourth day, everyfifth day, every sixth day, every week, and similarly in day incrementsup to 31 days or over. Alternatively, dosing can be continuous usingpatches, i.v. administration and the like.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active cupredoxin or variant, derivative or structuralequivalent thereof, with or without a cargo compound, which aresufficient to maintain therapeutic effect. Generally, the desiredcupredoxin or variant, derivative or structural equivalent thereof isadministered in an admixture with a pharmaceutical carrier selected withregard to the intended route of administration and standardpharmaceutical practice.

In one aspect, the cupredoxin or variant, derivative or structuralequivalent thereof is delivered as DNA such that the polypeptide isgenerated in situ. In one embodiment, the DNA is “naked,” as described,for example, in Ulmer et al., (Science 259:1745-1749 (1993)) andreviewed by Cohen (Science 259:1691-1692 (1993)). The uptake of nakedDNA may be increased by coating the DNA onto a carrier, e.g.,biodegradable beads, which are then efficiently transported into thecells. In such methods, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacterial and viralexpression systems. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.See, e.g., WO90/11092, WO93/24640, WO 93/17706, and U.S. Pat. No.5,736,524.

Vectors, used to shuttle genetic material from organism to organism, canbe divided into two general classes: Cloning vectors are replicatingplasmid or phage with regions that are essential for propagation in anappropriate host cell and into which foreign DNA can be inserted; theforeign DNA is replicated and propagated as if it were a component ofthe vector. An expression vector (such as a plasmid, yeast, or animalvirus genome) is used to introduce foreign genetic material into a hostcell or tissue in order to transcribe and translate the foreign DNA,such as the DNA of a cupredoxin. In expression vectors, the introducedDNA is operably-linked to elements such as promoters that signal to thehost cell to highly transcribe the inserted DNA. Some promoters areexceptionally useful, such as inducible promoters that control genetranscription in response to specific factors. Operably-linking acupredoxin and variants and derivatives thereof polynucleotide to aninducible promoter can control the expression of the cupredoxin andvariants and derivatives thereof in response to specific factors.Examples of classic inducible promoters include those that areresponsive to α-interferon, heat shock, heavy metal ions, and steroidssuch as glucocorticoids (Kaufman, Methods Enzymol. 185:487-511 (1990))and tetracycline. Other desirable inducible promoters include those thatare not endogenous to the cells in which the construct is beingintroduced, but, are responsive in those cells when the induction agentis exogenously supplied. In general, useful expression vectors are oftenplasmids. However, other forms of expression vectors, such as viralvectors (e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses) are contemplated. In addition, the peptides ofthe present invention, including in one embodiment, p18, may be used asa vector to selectively deliver therapeutic compounds into cancer cellsor tumors.

Vector choice is dictated by the organism or cells being used and thedesired fate of the vector. In general, vectors comprise signalsequences, origins of replication, marker genes, polylinker sites,enhancer elements, promoters, and transcription termination sequences.

Kits Comprising Cupredoxin, or Variant Derivative Or StructuralEquivalent Thereof

In one aspect, the invention provides regimens or kits comprising one ormore of the following in a package or container: (1) a pharmacologicallyactive composition comprising at least one cupredoxin or variant,derivative or structural equivalent thereof, (2) an additionalchemopreventive drug, (3) apparatus to administer the biologicallyactive composition to the patient, such as a syringe, nebulizer etc.

When a kit is supplied, the different components of the composition maybe packaged in separate containers, if appropriate, and admixedimmediately before use. Such packaging of the components separately maypermit long-term storage without losing the active components'functions.

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampoules may contain lyophilized cupredoxin andvariants, derivatives and structural equivalents thereof, or buffersthat have been packaged under a neutral, non-reacting gas, such asnitrogen. Ampoules may consist of any suitable material, such as glass,organic polymers, such as polycarbonate, polystyrene, etc., ceramic,metal or any other material typically employed to hold similar reagents.Other examples of suitable containers include simple bottles that may befabricated from similar substances as ampoules, and envelopes, that maycomprise foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, bottles, syringes, or thelike. Containers may have a sterile access port, such as a bottle havinga stopper that can be pierced by a hypodermic injection needle. Othercontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to be mixed.Removable membranes may be glass, plastic, rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audiotape, flash memory device etc. Detailedinstructions may not be physically associated with the kit; instead, auser may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

Modification of Cupredoxin, Cupredoxin Entry Domains and Variants,Derivatives and Structural Equivalents Thereof

Cupredoxin or variant, derivative or structural equivalents thereof maybe chemically modified or genetically altered to produce variants andderivatives as explained above. Such variants and derivatives may besynthesized by standard techniques. Cupredoxin entry domains may besimilarly modified.

In addition to naturally-occurring allelic variants of cupredoxin,changes can be introduced by mutation into cupredoxin coding sequencethat incur alterations in the amino acid sequences of the encodedcupredoxin that do not significantly alter the ability of cupredoxin toinhibit the development of premalignant lesions. A “non-essential” aminoacid residue is a residue that can be altered from the wild-typesequences of the cupredoxin without altering pharmacologic activity,whereas an “essential” amino acid residue is required for suchpharmacologic activity. For example, amino acid residues that areconserved among the cupredoxins are predicted to be particularlynon-amenable to alteration, and thus “essential.”

Amino acids for which conservative substitutions that do not change thepharmacologic activity of the polypeptide can be made are well known inthe art. Useful conservative substitutions are shown in Table 3,“Preferred substitutions.” Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the invention so long as the substitution doesnot materially alter the pharmacologic activity of the compound.

TABLE 3 Preferred substitutions Preferred Original residue Exemplarysubstitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln,Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L)Norleucine, Ile, Val, Met, Ala, Ile Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu Norleucine

Non-conservative substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge, (3) hydrophobicity, or (4) the bulk of the side chain of thetarget site can modify the pharmacologic activity. Residues are dividedinto groups based on common side-chain properties as denoted in Table 4.Non-conservative substitutions entail exchanging a member of one ofthese classes for another class. Substitutions may be introduced intoconservative substitution sites or more specifically into non-conservedsites.

TABLE 4 Amino acid classes Class Amino acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain conformation Gly, Proaromatic Trp, Tyr, Phe

The variant polypeptides can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis (Carter,Biochem J. 237:1-7 (1986); Zoller and Smith, Methods Enzymol.154:329-350 (1987)), cassette mutagenesis, restriction selectionmutagenesis (Wells et al., Gene 34:315-323 (1985)) or other knowntechniques can be performed on the cloned DNA to produce the cupredoxinvariant DNA.

Known mutations of cupredoxins can also be used to create variantcupredoxin to be used in the methods of the invention. For example, theC112D and M44KM64E mutants of azurin are known to have cytotoxic andgrowth arresting activity that is different from the native azurin, andsuch altered activity can be useful in the treatment methods of thepresent invention.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof.

EXAMPLES Example 1. Effect of Peptide p28 on DMBA-Induced MammaryLesions in the MMOC Model

The mouse mammary gland organ culture (MMOC) model allows evaluatingefficacy of potentially chemopreventive agents against development ofmammary alveolar lesions (MAL) or mammary ductal lesions (MDL) inresponse to DMBA. DMBA under appropriate incubation conditions formeither MAL or MDL based on the hormonal milieu in the medium. Hawthorneet al., Pharmaceutical Biology 40: 70-74 (2002); Mehta et al., J. Natl.Cancer Inst. 93: 1103-1106 (2001). Estrogen and progesterone-treatedglands in culture develop ductal lesions whereas aldosterone andhydrocortisone-treated glands form estrogen and progesterone-independentalveolar lesions. Mammary glands not exposed to a carcinogen orchemopreventive agent, undergo structural regression in the absence ofgrowth-promoting hormones, whereas treatment with DMBA for the 24-hrperiod between days 3 and 4 prevents the regression of structures causedby deprivation of hormones. It is assumed that this is because theglands have lost normal hormonal responsiveness and now have alteredtheir course of development. Generating mammary adenocarcinoma bytransplanting transformed cells into syngeneic mice has proved thepremalignant preneoplastic nature of these unrepressed areas.

The thoracic pair of mammary glands was excised aseptically from eachBalb/c mouse, and the glands were divided into several groups. Theeffects of p28 were evaluated at 4 different dilutions in the medium.Carcinogen treated glands without the test agent served as a measure todetermine percent incidence in the absence of a chemopreventive agent.An additional control was included to serve as a positive control forchemoprevention. Azurin was included in the medium at 50 μg/mlconcentration. For alveolar lesions (MAL) stained glands were evaluatedfor the incidence of lesions (glands containing any lesions as comparedto total number of glands in a given treatment group). For the ductallesions (MDL) similar protocol was adapted, however, as indicated belowin the methods section the hormonal combination is different foralveolar and ductal lesions. The glands were fixed in formalin and thenprocessed for histopathology. The sections are stained with cosin andhematoxelene and evaluated under microscope. Here the multiplicity ofductal lesions between the control and the treatment groups arecompared.

Organ Culture Procedure.

The experimental animals used for the studies were young, virgin BALB/cfemale mice 3 to 4 weeks of age obtained from Charles River, Wilmington,Mass. The mice were treated daily by subcutaneous injections with 1 μgestradiol-17β+1 mg progesterone for 9 days. This treatment is aprerequisite inasmuch as animals not pretreated with steroids fail torespond to hormones in vitro. The entire culture procedure is describedin detail. Jang et al., Science 275:218-220 (1997); Mehta, Eu. J. Cancer36:1275-1282 (2000); Mehta et al., J. Natl. Cancer Inst. 89:212-219(1997); Mehta et al., J. Natl. Cancer Inst. 93:1103-1106 (2001).

Briefly, the animals were killed by cervical dislocation, and thethoracic pair of mammary glands were dissected out on silk rafts andincubated for 10 days in serum free Waymouth MB752/1 medium (5-glands/5ml/dish). The medium was supplemented with glutamine, antibiotics(penicillin and streptomycin 100 units/ml medium) and growth-promotinghormones, 5 μg insulin (I), 5 μg prolactin (P), 1 μg aldosterone (A) and1 μg hydrocortisone (H) per ml of medium for the protocol to inducemammary alveolar lesions (MAL). For induction of ductal lesions (MDL),the medium contained 5 μg/ml, 5 μg/ml P, 0.001 μg/ml estradiol 17β and 1μg/ml progesterone (Pg). Mehta et al., J. Natl. Cancer Inst.93:1103-1106 (2001). The carcinogen, DMBA (2 μg/ml) was added to themedium between days 3 and 4. For the present study, DMBA was dissolvedin DMSO at a final concentration of 4 mg/ml, and 50 μg I was added to100 ml medium resulting in 2 μg/ml final concentrations. The controldishes contained DMSO as vehicle.

On day 4, DMBA is removed from the medium by rinsing the glands in freshmedium and transferring them to new dishes containing fresh mediumwithout DMBA. After 10 days of incubation, the glands were maintainedfor another 14 days in the medium containing only 1 (5 μg/ml). Duringthe entire culture period, the glands were maintained at 37° C. under95% O₂ and 5% CO₂ environment. The chemopreventive agent was included inthe medium during the first ten days of growth-promoting phase. The testpeptide p28 was evaluated at 4 concentrations ranging from 12.5 μg/ml to100 μg/ml. Azurin was evaluated at 50 μg/ml in the medium. The peptidewas dissolved in sterile water and filtered prior to use. The medium waschanged three times per week (Monday, Wednesday and Friday). At the endof the exposure, the glands were fixed in formain. Results were analyzedby Chi-square analysis and Fisher's Exact Test.

Morphometic Analysis of MAL.

For examination of MAL, the glands were stained in alum carmine, andevaluated for the presence of the lesions. The glands were scored forthe presence or absence of mammary lesions, severity of lesions pergland, and toxicity of the agent. The glands stored in xylene wereevaluated for the presence or absence, incidence, and severity ofmammary lesions for each gland under a dissecting microscope. Mammaryglands were scored as positive or negative for mammary lesions, and thepercent incidence was determined as a ratio of glands exhibiting lesionsand the total number of glands in that group. Dilation of ducts ordisintegration of mammary structure because of treatment withchemopreventive agent was considered a toxic effect. The data weresubjected to statistical analysis for the incidence to determine theeffectiveness of the potential chemopreventive agents.

FIG. 1A shows a representative photograph of alveolar lesions in a DMBAtreated gland and its comparison with a gland that was treated with DMBAalong with a chemopreventive agent. The effects of p28 on thedevelopment of alveolar lesion are shown in FIGS. 1B-1F and summarizedin FIG. 2. The peptide p28 inhibited MAL formation by 67% at 25 μg/mlconcentration. Increasing concentration further up to 100 μg/ml did notenhance the efficacy of the peptide. The comparison of the peptide withazurin indicated that p28 was as effective as azurin for MALdevelopment. Azurin at 50 μg/ml concentration resulted in a 67%inhibition. Statistical analyses indicated that the effect of p28 wasstatistically significant compared to DMBA control at concentrationsgreater than 12.5 μg/ml (p<0.01, Fisher's Exact Test; Chi Squareanalysis).

Histopathological Evaluation of MDL.

For MDL, the glands were processed for histopathological evaluations.The glands were sectioned longitudinally into 5-micron sections andstained with eosin hematoxeline. The longitudinal section of each glandwas divided into several fields and each field was evaluated for ductallesions. Mehta et al., J. Natl. Cancer Inst. 93:1103-1106 (2001).Briefly, the entire gland is evaluated under the scope; smaller glandswill have fewer total fields as compared to larger glands. Thus, eachgland will have variable number of fields. Often the number of sectionsthrough the ducts also varies greatly from gland to gland. This resultsin the variable number from group to group. Fields containing ductalhyperplasia or atypia were determined and were compared with totalnumber of field evaluated for each gland. No discrimination is madebetween the hyperplasia or atypia and severely occluded glands. Anyfield containing any of these histological patterns was consideredpositive for the lesion. The treatment groups were compared with thecontrols for the severity and percent inhibition was calculated.

FIG. 3 shows a representative ductal lesion. DMBA induces ductal lesionsvarying from hyperplasia, atypia to complete occlusion of the ducts. Aratio of ductal lesions/total number of ductal sections was determined.Again, 12.5 μg/ml concentration of p28 suppressed only 15% of the MDLformation. However, at 25 μg/ml there was a significant inhibition ofthe lesions comparable to that observed with 50 μg/ml azurin. Theefficacy of p28 at concentrations greater than 12.5 μg/ml wasstatistically significant (p<0.01, Fishers Exact Test). These resultsare summarized in FIG. 4. Often effects of chemopreventive agents can bedifferentiated between the MAL and MDL. For example tamoxifen inhibitedthe development of MDL but not MAL. It is interesting to note thatazurin and p28 inhibited both estrogen and progesterone-dependent ductallesions as well as independent alveolar lesions.

This example indicates that both p28 and azurin can prevent thedevelopment of precancerous lesions in breast tissue. Thus, p28 andazurin may be used as chemopreventive agents in mammalian patients.

Example 2. Selective Penetration of Cancer Cells by Cupredoxins andDerivative Peptides as Potential Vectors for Gene Delivery

Azurin, a member of the cupredoxin family of proteins, isolated fromPseudomonas aeruginosa, enters cancer cells and induces a p53-mediatedapoptosis in vitro and in vivo. The selectivity of penetration ofcationic and anionic cupredoxins and derived peptides as potentialvectors for gene delivery was evaluated. The following cupredoxins weretested: azurin (14 kDa, pI 5.7), rusticyanin (17 kDa, pI 8.0), andplastocyanin (11 kDa, pI 5.4). The results indicated that azurin had themost selective penetration.

25 amino acid (a.a.) fragments of azurin (azu) were synthesized andevaluated for their penetration into a variety of cancer andhistologicaly matched normal cells. Confocal microscopic and flowcytometric (FACS) analysis demonstrated that an 18 amino acid (1.7 kDa,azu 50-67) fragment (p18) labeled with Alexafluor 568 (800 Da)selectively penetrated human melanoma (Mel-2,7,29), breast (MCF-7),ovarian (SK-OV3), pancreatic (CAPAN-2), glioblastoma (LN-229),astrocytoma (CCF-STTG1), prostate (LN-CAP), and kidney (ACHN-CRL1611)cell lines, but not their respective controls. LDH release and hemolysisassays showed that p18 did not disrupt cancer cell membrane structureduring penetration or produce hemolysis of human erthrocytes, suggestingthat p18 penetrates human cancer cells without disrupting membranestructure. Pretreatment of Mel-2 cells with specific inhibitors of cellinternalization (cytochalasin D; inhibition of actin polymerization,taxol; inhibition of microtubule depolymerization, chlorpromazine;inhibition of clathrin-mediated endocytosis, sodium azide; metabolicinhibition, or staurosporine; cell cycle inhibition) had a negligibleeffect on the penetration of p18. However, incubation of Mel-2 cellswith nystatin (caveolae formation inhibitor) and brefeldin A (golgiapparatus disrupter) significantly inhibited the penetration of p18,suggesting that endocytic processes may, in part, be involved in thepenetration of p18. Imaging of p18 labeled with an infrared dye (λ_(em)800 nm) in athymic mice bearing xenografted melanoma tumors clearlydemonstrated selective uptake in primary s.c. tumors and distant organmetastases without accumulating in normal organs and tissues. As such,the peptides of the present invention, including in one embodiment, p18appear to have significant utilization as a non-viral vector for gene(or any DNA/RNA fragment) therapy.

Example 3—Plasmid Constructions

Plasmids expressing fusion glutathione S-transferase (GST)-truncatedwt-azurin (azu) derivatives were constructed by a polymerase chainreaction using proofreading DNA polymerase. FIG. 5 shows a schematicrepresentation of various truncated wt-azurin constructs. For pGST-azu36-128, an amplified PCR fragment was introduced into the BamHI andEcoRI sites of the commercial GST expression vector pGEXSX (AmershamBiosciences, Piscataway, N.J. 08855). The fragment was amplified withpUC19-azu as a template and primers, 5′-CGGGATCC CCG GCA ACC TEC CGA AGAACG TCA TGGGC-3′ (SEQ ID NO: 78) and 5′-CGGAATTC GCA TCA CTT CAG GGT CAGGG-3′ (SEQ ID NO: 79), where the additionally introduced BamHI and EcoRIsites are underlined respectively. Carboxyl-terminus truncation of azugene was cumulatively performed by introducing a stop codon usingQuickChange site-direct mutagenesis kit (Stratagene, La Jolla, Calif.92037).

For pGST-azu 36-50, pGST-azu 36-77 and pGST-azu 36-89, stop codons wereintroduced into Ser51, Ser78, and Gly90, respectively. The plasmidcarrying pGST-azu 36-128 was used as template DNA. Three sets ofoligonucleotides for site-direct mutagenesis are shown as follows. ForpGST-azu 36-50:5′-GGC CAC AAC TGG GTA CTG TGA ACC GCC GCC GAC ATG CAG-3′(SEQ ID NO: 80), and 5′-CTG CAT GTC GGC GGC GGT TCA CAG TAC CCA GTT GTGGCC-3′ (SEQ ID NO: 81). For pGST-azu 36-77: 5′-CCT GAA GCC CGA CGA CTGACG TGT CAT CGC CCA CAC C-3′ (SEQ ID NO: 82) and 5′-GGT GTG GGC GAT GACACG TCA GTC GTC GGG CTT CAG G-3′ (SEQ ID NO: 83). For pGST-azu 36-89:5′-CCA AGC TGA TCG GCT CGT GAG AGAAGG ACT CGG TGA CC-3′ (SEQ ID NO: 84),and 5′-GGT CAG CGA GTC CTT CTC TCA CGA GCC GAT CAG CTT GG-3′ (SEQ ID NO:85). The plasmids pGST-azu 50-77 and pGST-azu 67-77 were generated byPCR using pGST-azu 36-77 as a template DNA.

Amplified PCR fragments, azu 50-77 and azu 67-77, were obtained usingforward primers 5′-CGGGATCC TGA GCA CCG CCG CCG ACA TGC AGG G-3′ (SEQ IDNO: 86) and 5′-CGGGATCC CCG GCC TGG ACA AGG ATT ACC TGA AGC CCG-3′ (SEQID NO: 87), where the additionally introduced BamHI site is indicated byunderlining. The reverse primer, 5′-CGGAATTC GCA TCA CTT CAG GGT CAGGG-3′, was utilized in both cases (SEQ ID NO: 88).

The plasmid carrying gst-azu 50-77 was used for generating pGST-azu50-66 by introduction of a stop codon in Gly67 using oligonucleotides asfollows: 5′-GAC GGC ATG GCT TCC TGA CTG GAC AAG GAT TAC C-3′ (SEQ ID NO:89), and 5′-GGT AAT CCT TGT CCA GTC AGG AAG CCA TGC CGTC-3′ (SEQ ID NO:90). The green fluorescent protein gene (gfp) encoding the greenfluorescent protein was also amplified by PCR. Forward and reverseprimers used were 5′-CGGGATCC CCA TGG TGA GCA AGGGCG-3′ (SEQ ID NO: 91)and 5′-CGGAATTC CTT GTA CAG CTC GTC CAT GCC G-3′ (SEQ ID NO: 92)containing BamHI and EcoRI sites at the 5′ end of each oligonucleotides.The resultant PCR fragment was ligated into the pGEXSX vector forcreating pGST-GFP. For the preparation of plasmid DNA carryinggst-gfp-azu 50-77, the azu 50-77 gene was amplified by PCR with pGST-azu50-77 as a template and primers 5′-CCGCTCGAG CCT GAG CAC CGC CGC CATGCAGGG-3′ (SEQ ID NO: 93) and 5′-TTTTCCTTTTGCGGCCGC TCA GTC GTC GGG CTT CAGGTA ATC C-3′ (SEQ ID NO: 94), where the introduced Xho I and Not I sitesare underlined respectively. Purified azu 50-77 fragment was introducedinto pGST-GFP at Xho 1 and Not 1 unique restriction enzyme sites

Example 4—Purification of Proteins

Wt-azurin and M44KM64E mutant azurin were prepared and purified asdescribed by Yamada, T. et al. Proc. Natl. Acad. Sci. USA, vol. 101, pp.4770-75 (2004), and in copending U.S. patent application Ser. No.10/720,603, the contents of which are incorporated by this reference.Briefly, the wt-azurin gene was amplified by PCR according to the methoddescribed by Kukimoto et al., FEBS Lett, vol. 394, pp 87-90 (1996). PCRwas performed using genomic DNA from P. aeruginosa strain PAO1 as atemplate DNA.

The amplified DNA fragment of 545 bp, digested with HindIII and Pstl,was inserted into the corresponding sites of pUC19 so that the azuringene was placed down-stream of the lac promoter to yield an expressionplasmid pUC19-azuA. E. coli JM109 was used as a host strain forexpression of the azurin gene. The recombinant E. coli strain wascultivated in 2YT medium containing 50 μg ml⁻¹ ampicillin, 0.1 mM IPTG;and 0.5 mM CuSO₄ for 16 h at 37° C. to produce azurin.

For preparation of the M44KM64E mutant azurin, site-directed mutagenesisof the azurin gene was performed using a QuickChange site-directedmutagenesis kit (Stratagene, La Jolla, Calif.). Mutations were confirmedby DNA sequencing.

Plasmid DNA, pET9a carrying the rus gene encoding the cupredoxinrusticyanin from Acidithiobadilus ferrooxidans, was obtained from Dr.Kazuhiko Sasaki, Central Research Institute of Electric Power Industry,Chiba, Japan.

Rusticyanin was isolated from E. coli BL21 (DE3) harboring the rus geneusing the method of Sasaki, K., et al. Biosci. Biotechnol. Biochem.,vol. 67, pp. 1039-47 (2003) with some modifications. Briefly, aceticacid buffer (pH 4.0) and CM-Sepharose (Sigma Chemicals, St. Louis, Mo.63178) were used instead of beta-alanin buffer (pH 4.0) and TSK-gelCM-650 column (Tosoh Bioscience, LLC, Montgomeryville, Pa. 18936). Twoother purified cupredoxins, plastocyanin from Phormidium laminosum andpseudoazurin from Achromobacter cycloclastes were obtained from Dr.Beatrix G. Schlarb-Ridley, University of Cambridge, UK and Dr.Christopher Dennison, University of Newcastle Upon Tyne, UK,respectively.

All recombinant GST-fusion derivatives were purified as follows: E. coliBL21 cells were used as the host strain. After induction with 0.4 mMIPTG at early log phase of growth in L broth, GST-fusion proteins werepurified from cell extracts by using Glutathione Sepharose 4B affinitychromatography and Sephadex 75 gel-filtration column with PBS (AmershamBiosciences, Piscataway, N.J. 08855). Purified proteins, wt azurin andGST-derivatives or other cupredoxins, labeled with ALEXA FLUOR®(Molecular Probes, Inc., Eugene, Oreg. 97402) were isolated according tomanufacturer's instructions. Unbound free fluorescent chemical wasremoved by gel-filtration column.

Example 5—Cell Cultures

J774 and UISO-Mel-2 cells (available from Frederick Cancer Research andDevelopment Center, Frederick, Md. U.S.A.) were cultured as described inYamada, T. et al. Infect. Immun. vol. 70, pp. 7054-62 (2002); Goto, M.,et al. Mol. Microbiol. vol. 47, pp. 549-59 (2003); and Yamada, T., etal. Proc. Natl. Acad. Sci. USA vol. 99, pp. 14098-103 (2002), thecontents of which are incorporated by reference. Human normal fibroblastcells (stock culture collection of the Department of Surgical Oncology,University of Illinois at Chicago (UIC), Chicago) were cultured in MEMwith Eagle's salt containing 2 mM L-glutamine, 0.1 mM MEM essentialamino acids and supplemented with 10% heat inactivated fetal bovineserum, 100 Units/ml penicillin and 100 μg/ml streptomycin. MCF-7 andMOF-10F cells were cultured as described in Punj et al. Oncogene23:2367-78 (2004).

Example 6—Co-culture of J774, UISO-Mel-2 and Fibroblast Cells andConfocal Microscopy

J774, UISO-Mel-2, and fibroblast cells were cultured on individual coverslips. After overnight incubation, the cells were washed with freshmedia and all three cell lines were placed on a culture dish containing200 μg/ml of wt-azurin conjugated with ALEXA FLUOR® 568. The cells werethen incubated for 0.5 or 3.5 h at 37° C. under 5% CO₂.

For preparation of microscope samples, cells were cultured oncover-slips overnight at 37° C. Cultured cells were placed at 37° C. or4° C. for 2 h before protein treatment. Pre-warmed 37° C. fresh media orice-cold 4° C. fresh media were mixed with red-fluorescent (labeled withALEXA FLUOR® 568) cupredoxins or GST-fusion derivatives, and incubatedwith the cells. The cells were washed with PBS, and fixed with methanolat −20° C. for 5 min. After washing with PBS twice and the addition ofmounting media containing 1.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI)for staining nuclei (VECTASHILD, Vector, Burlingame, Calif.), imageswere taken by a confocal microscope.

Example 7—Entry of Cupredoxins into J774 Cells

Wt-azurin, its mutant variant M44KM64E, plastocyanin, pseudoazurin andrusticyanin were incubated with J774 cells as in Example 6 and the cellsexamined using confocal microscopy. In these experiments, thecupredoxins were conjugated with ALEXA FLUOR® 568 to fluoresce red andincubated with the J774 cells for 1 hr at 37° C. at a concentration of200 μg/ml, and in a separate experiment wild type azurin and rusticyaninwere incubated with J774 cells for 1 hr at 37° C. at a concentration ofabout 6 to 7 μM. The nucleus was stained blue with DAPI. A controlwithout the proteins was maintained. In all cases, the cupredoxins wereseen to enter into the cytosol of J774 cells. In similar experiments,auracyanin A and B enter preferentially to MCF7 cancer cells and notnon-cancerous control cells.

Example 8—Entry of Wt-azurin and Rusticyanin into Various Cell Types

Wt-azurin exhibits a reduced cytotoxic activity towards MCF-10F cells ascontrasted with the MCF-7 cells. Punj et al. Oncogene 23:2367-2378(2004). J774, peritoneal macrophages, mast cells, human breast cancerMCF-7 and human normal epithelial MCF-10F cells (stock culturecollection of the Department of Surgical Oncology, University ofIllinois at Chicago (UIC), Chicago) were treated and examined as inExample 5 and tested to determine if wt-azurin could enter such cells.

Wt-azurin was internalized in J774 cells during 45 mm incubation.However, it was internalized very inefficiently in peritonealmacrophages or mast cells. Even after 6 hr incubation, such cells showedonly limited entry. Similarly, while wt-azurin entered the breast cancerMCF-7 cells efficiently, it showed an extremely reduced rate of entry inthe normal mammary MCF-10F cells.

Alexa Fluor®-conjugated azurin entered efficiently in UISOMel-2 andMCF-7 cancer cells but not in the normal mammary MCF 10A1 cells. AlexaFluor®-conjugated rusticyanin, however, not only entered the cytosol ofUISO-Mel-2 and MCF-7 cancer cells, but also in the normal MCF 10A1cells. Unlike in the cancer cells where rusticyanin was evenlydistributed in the cytosol, in MCF10A1 cells, much of the rusticyaninwas sequestered in the perinuclear space surrounding the nucleus.

Example 9—Wt Azurin-Mediated Cytotoxicity and Growth Inhibition

To further assess the specificity of entry of wt-azurin in variouscells, the entry of Alexa fluor-conjugated wt-azurin in J774, UISO-Mel-2and normal fibroblast cells was determined during incubation at 37° C.for 30 min and 3.5 hr. Wt-azurin was seen to enter rapidly in J774 andUISO-Mel-2 cells in 30 mm; very little wt-azurin was seen in the cytosolof fibroblasts during this period. After 3.5 hr of incubation, onlysmall amounts of wt-azurin were found in the fibroblasts.

A 3(4,5 dimethylthiazol-2-yl-2,5 tetrazolium bromide)(MTT) assay wasperformed for the measurement of the cytotoxicity of wt-azurin asdescribed by Yamada, T., et al. Infect, Immun. 70:7054-62 (2002), Goto,M., et al. Mol. Microbiol. 47:549-59 (2003), and in co-pending U.S.patent application Ser. No. 10/720,603, filed Nov. 24, 2003, thecontents of which are incorporated by reference. FIG. 1(b) shows thatsignificant wt-azurin-mediated cytotoxicity was observed only with J774and UISO-Mel-2 cells during 24 hr incubation.

M44KM64E mutant azurin showed very little apoptosis-inducing activity inJ774 cells but at 1 mg/ml concentration significantly inhibited (about95%) cell cycle progression at the G₁ to S phase. Cell cycle progressionwas analyzed by flow cytometry, as described by Hiraoka, Y. et al.,Proc. Natl. Acad. Sci. USA, vol. 101:6427-32 (2004) and Yamada, T. etal. Proc. Natl. Acad. Sci. USA 101:4770-75 (2004), the contents of whichare incorporated by reference. FIG. 1(a) shows that when the fibroblastswere treated with 500 μg/ml or 1 mg/ml of M44KM64E mutant azurin, theextent of inhibition of cell cycle progression was about 20%.

Example 10—Microinjection of Wt-azurin into Fibroblast and MCF-10F Cells

Wt-azurin was microinjected into fibroblast and MCF-10F cells as usingthe method described by Punj, V., et al., Oncogene 23:2367-78 (2004).Cells were examined for induction of apoptosis, leading to nuclear DNAcondensation and fragmentation. Significant nuclear DNA (labeled bluewith DAPI) condensation and fragmentation were observed in microinjectedsingle cells after 5 hr incubation with wt-azurin, but not during a 30min. incubation with azurin.

Example 11—Internalization of Wt-azurin Fusion Derivatives at 37° C.

A series of GST fusions of wt-azurin truncated at both the N- and theC-terminal were prepared and purified as in Example 1 (FIGS. 2(a) and2(b). Using ALEXA FLUOR® 568 conjugated wt-azurin, GST and GST-azufusion derivatives, internalization in J774 cells at 37° C. during 1 hrincubation was examined using the method described in Example 5. Thenucleus was stained blue with DAPI.

While wt-azurin was internalized, GST remained at the periphery of thecells and was not internalized. GST-azu 36-128 and GST-azu 36-89 wereinternalized, as was GST-azu 36-77. Further truncations, however,demonstrated that while GST-azu 50-77 was internalized, GST-azu 36-50was highly inefficient and appeared to form clumps on the surface.

Example 12—Internalization of Azurin Fusion Derivatives at 4° C.

Internalization of wt-azurin and the GST-azu fusion derivatives in J774cells incubated at 4° C. was examined. At 4° C., internalization ofwt-azurin inside J774 cells during 1 hr incubation was severelyimpaired. Similar impairment was also seen with GST-azu 36-128 andGST-azu 36-89. The shorter GST-azu 36-77, GST-azu 50-77, GST-azu 50-66and GST-azu 67-77 demonstrated severe impairment of internalization at4° C.

Example 13—Energy-Dependent Internalization of the GST-GFP-azu 50-77Fusion Protein in J774 and Melanoma UISO-MeI-2 Cells

GST was fused with GFP to make a GST-GFP fusion derivative.Additionally, azu 50-77 was fused to the GST-GFP (M_(r) 53 kDa) fusionprotein (FIG. 6(a)). The mobility of the purified GST, GST-GFP andGST-GFP-azu 50-77 fusion derivatives was examined on SDS-PAGE (FIG.6(b)). Detection was by Coomassie Blue staining and Western blottingusing anti-azurin antibody (FIG. 6(c))

Flow cytometric determination of J774 cells treated with varyingconcentrations of GST-GFP showed that this protein does bind to J774cells. Flow cytometric separation of J774 cells treated with increasingconcentrations of GST-GFP-azu 50-77 fusion protein demonstratedsignificantly reduced fluorescence than GST-GFP alone (FIG. 7). It is tobe noted that internalization of GFP in mammalian cells is known to leadto loss of fluorescence. This reduction of fluorescence is also apparentwhen J774 cells are treated with 200 μg/ml of GST-GFP-azu 50-77 fusionprotein and incubated for increasing periods of time at 37° C.

To determine if there is any difference in the binding andinternalization profile of GST-GFP and GST-GFP-azu 50-77, both J774 andUISO-Mel-2 cells were incubated with GST-GFP and GST-GFP-azu 50-77 at37° C. and at 4° C. The green fluorescence was localized using confocalmicroscopy. In J774 cells, GST-GFP fusion protein bound to the surfaceand was not internalized both at 37° C. and at 4° C. In contrast,GST-GFP-azu 50-77 was found to be internalized at 37° C., but not at 4°C. In UISO-Mel-2 cells, the GST-GFP fusion protein was retained on thesurface both at 37° C. and at 4° C. In contrast, similar to J774 cells,GST-GFP-azu 50-77 fusion protein was seen to be internalized at 37° C.but not at 4° C.

Example 14—Wt-azurin Entry into Mammalian Cells by a Cell MembranePenetration and an Endocytic Mechanism

If wt-azurin entry is solely dependent on receptor-mediated endocytosis,it could be blocked by protonophore carbonyl cyanidem-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler of energygeneration, or preincubation with unlabeled azurin or other cupredoxinsthat block the receptors. J774 and UISO-MeI-2 cells were incubated withthe cupredoxins at 10 fold excess concentration for 2 hr at 4° C., thecells washed thoroughly to remove the cupredoxins, and incubated withALEXA FLUOR® 568-conjugated azurin for 1 hr at 37° C. There was as muchinternalized azurin as in cells not treated with the cupredoxins. Theeffects of cytochalasin D (available from Sigma-Aldrich, St. Louis, Mo.63195), a known inhibitor of receptor-mediated endocytosis that disruptsthe cellular microfilament network, and Brefeldin A (available fromSigma-Aldrich, St. Louis, Mo. 63195), which is known to disrupt theGolgi apparatus and inhibit classical vesicle-mediated secretion, werealso tested. CCCP at 20 pM concentration significantly reduced theuptake of azurin in UISO-MeI-2 cells as did 0.25 to 0.5 μM cytochalasinD. Brefeldin A, on the other hand, had no significant effect.

Example 15—Entry of a GST-PEDIII-azu 50-77 Fusion Derivative intoUISO-Mel-2 Cells

A GST-fusion of Pseudomonas aeruginosa exotoxin A domain III (PEDIII)was constructed as described by Hwang, J. et al., Cell 48:129-36 (1987);Reiter, Y. and Pastan, I., Trends Biotechnol. 16:513-20 (1998). ThisGST-PEDIII fusion derivative contained amino acids 381-613 of theexotoxin A. PEDIII is known to harbor ADP-ribosyl transferase activityand inhibits cellular protein synthesis in eukaryotic cells byinhibiting eukaryotic elongation factor 2.

Using PCR as described for the GST-GFP-azu 50-77, the azu 50-77 sequencewas introduced to the carboxyl end of the GST-PEDIII fusion protein(FIG. 8(a)). These two fusion proteins (GSTPEDIII and GST-PEDIII-azu50-77) were purified by glutathione-sepharose 4B column chromatographyas 52 and 54 kDa proteins (FIG. 8(b)). UISO-Mel-2 and normal fibroblast(FBT) cells were then incubated for 24 h at 37° C. with variousconcentrations of these proteins and the extent of cell death measuredby MTT assay as described in Example 9.

While GST-PEDIII demonstrated only low cytotoxicity, the GST-PEDIII-azu50-77 fusion protein had high cytotoxicity because of efficient entry toUISO-Mel-2 cells (FIG. 8(c)). In contrast, the fusion proteinsdemonstrated a low level of cytotoxicity towards the fibroblast cells.

Example 16—Destabilization of the α-helix in wt-Azurin has noSubstantial Effect on its Internalization in UISO-Mel-2 Cells

To examine if the α-helix plays a role in azurin entry, threehelix-destabilizing proline residues were introduced in positions 54, 61and 70 of wt-azurin (FIG. 6) and examined the entry of the full lengthA54PT61PK70P mutant azurin into UISO-Mel-2 cells. Single and doublemutations in these positions were also constructed and tested for entry.The A54PT61PK70P mutant azurin was prepared by site-directed mutagenesisof the azurin gene using the QuickChange site-directed mutagenesis kit(Stratagene, La Jolla, Calif.).

The mutants were incubated at 200 μg/ml with UISO-Mel-2 cells for 1 hrat 37° C., after which the fluorescence was localized by confocalmicroscopy. In all cases, the ALEXA FLUOR® 568-conjugated mutant azurinsentered UISO-Mel-2 cells. Similarly, when the GST-GFP-azu 50-77 fusionprotein, as well as its triple A54PT61PK70P azu mutant variant, wereexamined for entry in UISO-Mel-2 cells, no significant difference wasobserved.

Example 17—Entry of a GST-PEDIII-Rusticyanin Fusion Derivative intoUISO-Mel-2 Cells

A GST-fusion of Pseudomonas aeruginosa exotoxin A domain III (PEDIII)and was constructed as in Example 15. Using PCR as described for theGST-GFP-azu 50-77, full-length rusticyanin sequence was introduced tothe carboxyl end of the GST-PEDIII fusion protein. The fusion proteinwas purified by glutathione-sepharose 4B column chromatography.UISO-Mel-2 and FBT cells were then incubated for 24 h at 37° C. withvarious concentrations of the fusion protein and the extent of celldeath measured by MTT assays as described in Example 7.

The GST-PEDIII-rusticyanin fusion protein exhibited high cytotoxicityagainst UISO-Mel-2 cells (FIG. 9). In contrast, the fusion proteindemonstrated only a low level of cytotoxicity towards the FBT cells.

Example 18—Entry of p18 and p28 Into Human Cell Lines

Cell Culture and Cell Lines:

Human cancer and non-cancer (immortalized and non-immortalized) celllines were obtained from ATCC [lung cancer (A549 and NCI-H23adenocarcinoma), normal lung (CCD-13Lu), prostate cancers (DU145 andLN-CAP), normal prostate (CRL11611), breast cancer (MCF-7), normalbreast (MCF-10A), colon cancer (HCT116), normal colon (CCD33Co),fibrosarcoma (HT1080), and ovarian cancer (SK-OV3 adenocarcinoma)].Normal fibroblasts isolated from skin were established. Normal ovariancells (HOSE6-3) were donated by Dr. S. W. Tsao (University of HongKong). Melanoma lines (UISO-Mel-2, 23, 29) were established andcharacterized. All cells except UISO-Mel-2 were cultured in MEM-E(Invitrogen, Carlsbad, Calif.) supplemented with 10% heat-inactivatedfetal bovine serum (Atlanta Biological Inc., Lawrenceville, Ga.), 100units/ml penicillin and 100 μg/ml streptomycin at 37 C in 5% CO2 or air.

Proliferation Assays/Cell Growth:

Melanoma cells were seeded (four replicates) in flat bottom 24 wellplates (Becton Dickinson, Franklin Lakes, N.J.) at a density of 12×103cells/well. After 24 hrs media was changed and fresh p18, p28, azurin ora similar volume of media without peptide (eight replicates) added dailyfor 72 hr. Cells were then counted in a Beckman Coulter (Z 1 coulterparticle counter). Values represent the mean±SD of 4 replicates.

MITT Assay:

Melanoma cells were seeded at a density of 2000 cells/well inflat-bottomed 96 well plates (Becton Dickinson, Franklin Lakes, N.J.)and allowed to attach for 24 hrs. Freshly prepared peptide (10 μl) orculture medium was then added to each well. After 24 hrs, medium waschanged and p18, p28 or azurin added daily. After 72 hr incubation, 10μl of MTT reagent (Trevigen, Gaithersburg, Md.) was added to each well,the samples incubated for 3 hr, RT/sig 100 μl of detergent added to eachwell, and the samples incubated for an additional 3 hr at 37° C.Absorbance was measured with a SpectraMax 340 plate reader (MolecularDevices Corporation, Sunnyvale, Calif.) and percent change in theabsorbance at 570 nm in treated cells relative to untreated controlsdetermined. Values represent the mean±SD. Significance between controland treated groups was determined by Student's t-test.

Peptide Synthesis:

All azurin derived peptides including p18, Leu⁵⁰-Gly⁶⁷LSTAADMQGVVTDGMASG (SEQ ID NO. 25), p28 Leu⁵⁰-Asp⁷⁷LSTAADMQGVVTDGMASGLDKDYLKPDD (SEQ ID NO. 2), p18b Val⁶⁰-Asp⁷⁷VTDGMASGLDKDYLKPDD (SEQ ID NO. 34), MAP, Mastoparan-7, and poly arginine(Arg₈) (SEQ ID NO: 95) were synthesized by C S Bio, Inc. (Melo Park,Calif.). Peptides were received as lyophilized powder aliquoted andstored at −20° C. in air-tight desiccators. All peptides weresubsequently analyzed by mass spectrometry and reverse phase HPLCas >95% purity and mass balance.

Predictive Modeling for Azurin Peptides:

GENETYX software (ver. 6.1) was used to generate Robson structure modelsfor azurin derived peptides. Gamier, J., Osguthorpe, D. J., and Robson,B., J Mol Biol, 120: 97-120 (1978). The MAPAS Software was used topredict a given protein structure for strong membrane contacts anddefine regions of the protein surface that most likely form suchcontacts. Sharikov, Y. et al, Nat Methods, 5: 119 (2008). If a protein,i.e., azurin, has a membranephilic residue score (MRS)>3, membranephilicarea score (MAS)>60%, and coefficient of membranephilic asymmetry(K_(mpha))>2.5, there is a high probability that the protein has a truemembrane-contacting region.

Peptide/Protein Labeling:

Peptides were dissolved in 1 ml PBS mixed with Alexafluor 568 dye(Molecular Probes, Eugene, Oreg.) at a 1:2 protein:dye ratio, 100 μlsodium bicarbonate added, and the mixture incubated overnight at 4° C.with continuous stirring. Labeled peptide was separated from free dye bydialyzing against cold-PBS using Slide-A-Lyzerg Dialysis Cassettes 1000MWCO for p12 and 2000 MWCO for others (Pierce Biotechnology, Rockford,Ill.).

Cell Penetration/Confocal Analysis:

Cells were seeded on glass coverslips and allowed to attach overnight at37° C. under 5% CO₂. Cells were rinsed with fresh media and incubated at37° C. for 2 hrs in pre-warmed media containing Alexafluor 568 labeledazurin peptides (20 μM) or Arg8 (SEQ ID NO: 95) (5 μM), or media alone.Following incubation, coverslips were rinsed 3× with PBS, cells fixed in2.5% formalin for 5 min, and washed 2× in PBS, once in d.i. H₂O, andcoverslips mounted in media containing 1.5 μg/ml DAPI for nuclearcounter staining (VECTASHIELD® Vector Laboratories, Burlingame Calif.).Cellular uptake and distribution were photographed under an invertedconfocal laser scanning microscope (Model LC510, Carl Zeiss Inc.,Gottingen, Germany).

Peptide co-localization with lysosomes or mitochondria was determined byincubating cells growing on a glass coverslip for 2 hrs at 37° withAlexafluor 568 labeled azurin or peptides. Mitrotracker (MitroTracker®Green FM Invitrogen Corporation, Carlsbad, Calif.) or lysotracker(LysoTracker® Green DND-26 Invitrogen Corporation, Carlsbad, Calif.) wasadded (final concentration 1 μM) for the last 30 mins of incubation.Cells were rinsed 3× with PBS, fixed in 2.5% formalin for 5 mins, washed2× with PBS and incubated in 0.1% Triton-X100 in PBS for 15 min. Cellswere then incubated with 1 μg/ml rabbit anti-human golgin 97 oranti-human caveolin I (Abcam, Cambridge, Mass.) in PBS with 1% BSA.After 1 hr incubation at 4° C., coverslips were washed once with PBS,incubated 10 min in PBS containing Alexafluor 468 conjugated goatanti-rabbit antibody, washed 2× in PBS and once in d.i.H20. Coverslipswere then mounted in media containing 1.5 μg/mlDAPI for nuclear counterstaining. Colocalization (yellow) of Alexafluor 568 (red) and Alexafluor468 (green) was analyzed and photographed.

UISO-Mel-2 cells on coverslips were preincubated in MEM-E containing 100μg/ml heparin sulfate (Sigma-Aldrich, St. Louis, Mo.) for 30 min andp18, p28 or Arg₈ (SEQ ID NO: 95) added to bring the final concentrationto 20 μM. After 1 hr, coverslips were washed, fixed, and analyzed asdescribed above.

Cell Penetration by FFACS:

Cells (1.0×10⁶/500 μl PBS) were incubated for 2 hrs at 37° C. withAlexafluor 568 labeled p18 or p28 (20 μM), Arg₈ (SEQ ID NO: 95) (5 μM),or media alone, washed 3× in PBS, fixed in 2.5% formalin for 5 min,washed twice in PBS, resuspended in 200 μl PBS, and passed through ascreen to obtain a single cell suspension. Samples were analyzed with aMoFlo Cell Sorter (Dako, Glostrup, Denmark) λ_(ex) 568 nm and λ_(em) 603nm and the fold increase of the mean fluorescence intensity overbackground levels calculated. Results represent mean fluorescence ofthree separate experiments.

Entry Inhibitors:

UISO-Mel-2 cells (3×10⁵ per 300 μl), maintained in phenol red-,serum-free MEM-E at 37° C., were pretreated with inhibitors, including:Chloropromazine (inhibitor of clathrin-mediated endocytosis, 10 μg/ml,60 min); Amiloride (macropinocytosis inhibitor, 50 μM, 30 min); Nystatin(50 μg/ml, 30 min); Methyl-β-cyclodextrin (MβCD, 5 mM, 60 min); Filipin(inhibitor of caveolae-mediated endocytosis, 3 μg/ml, 60 min); Taxol(microtubule stabilizer, 20 μM, 30 min); Staurosporine (cell cycleinhibitor, 250 nM, 10 min); Sodium azide (metabolic inhibitor, 1 mM, 60min); Oauabain (ATPase-dependent Na+/K+ pump inhibitor, 50 mM, 60 min);Brefeldin A (BFA; Golgi apparatus disruptor, 100 μM, 60 min); Wortmannin(early endosome inhibitor, 100 nM, 30 min); Monensin (inhibits at lateendosome/lysosome, 10 μM, 60 min); Nocodazole (inhibits caveosomeformation, 10 μM, 60 min); Cytochalasin D (actin filament andmicrotubule disruptor, 5 μM, 30 min); Benzyl2-acetamido-2-deoxy-α-D-galactopyranoside (BnGalNac; O-linkedglycosylation inhibitor, 3 mM, 48 hrs); Tunicamycin (N-linkedglycosylation inhibitor, 20 μg/ml, 48 hrs); and Neuraminidase (cleavesialic acid residues from proteins, IU/ml, 30 min). Final concentrationswere derived from the dose response curves of individual inhibitors.Alexafluor 568 labeled p18 or p28 (20 μM) were then added, incubated for1 hr, and the cells washed, fixed and prepared for flow cytometricanalysis as described above.

Cell Membrane Toxcity Assays/LDH Leakage Assay:

An LDH leakage assay was performed according to the manufacturer'sinstructions (CytoTox-One, Promega, Wis.) with 100 μl of UISO-Mel-2cells (5×10³). Cells without peptides/proteins were used as a negativecontrol. Experiments were carried out in triplicate (data representmean±SEM).

Hemolysis Assay:

Human whole blood samples (2-3 ml) were centrifuged for 10 min at 100×g,and the pellets washed once with PBS and once with HKR buffer pH7.4(18). Cell pellets were then resuspended in HKR buffer to 4%erythrocytes, 50 μl transferred to a 1.5 ml tube with 950 μl ofpeptides, azurin (5, 50 and 100 μM) or 0.1% Triton X-100 in HRK bufferto completely disrupt the RBC membrane. MAP and Mastoparan7 (BachemCalifornia, Inc., Torrance, Calif.) were used as positive controls.After 30 min incubation at 37° C. with rotation, tubes were centrifugedfor 2 min at 1000×g, 300 μl of supernatants transferred to a 96-wellplate and absorbance recorded at 540 nm.

Kinetics of Entry:

UISO-Mel-2 cells (5×10⁵ cells) in 1.5 ml tubes were suspended in MEMEmedia without phenol red. Reactions were started by adding either Alexafluor 568-conjugated p18 at 0, 10, 20, 50, 100, 150 and 200 μM for 5,10, 15 and 20 sec., or Alexafluor 568-conjugated p28 at 1, 10, 25, 50,100, 150 and 200 μM for 30, 60, 90 and 120 see on ice. After incubation,1 ml of cold-PBS was added to the 250 μl reaction in mixture. Cells werecentrifuged twice at 600×g for 2 min at 4° C. At least 10,000 fixedcells were analyzed by flow cytometry in each reaction and theirbackground and relative fluorescence calculated.

¹²⁵I Labeling of Azurin and Competition Assays:

Peptide binding and entry was determined using a whole cell assay withUISO-Mel-2 cells in HEPES solution (50,000 cells/ml), were incubated for30 min at 37° C. with increasing concentrations (0-175 nM) ofradiolabeled a zurin in the presence/absence of 1000 fold excess ofunlabeled p18, p28, or azurin, then washed 3 times with ice cold PBS,and radioactively remaining in the cell pellet counted using a gammacounter. Radioactivity in cells incubated with ¹²⁵I azurin alone wasconsidered total binding; radioactivity in the presence of unlabeledazurin, p18, or p28 was considered nonspecific binding. Specific bindingwas determined by subtracting nonspecific binding from total binding andScatchard plots generated.

Example 19—Domain of p28 Responsible for Preferential Entry into CancerCells

Initial data from peptide-GST constructs defined amino acids 50-77 ofazurin as a PTD for cell penetration, which fits well with structuralevidence for an-helical region encompassing residues 54-67 of azurinstabilizing the azurin molecule. Confocal analyses initially suggestedthat p28 and p18 of p28/azurin (FIG. 10A and B) penetrated humanmelanoma, prostate, lung, breast and ovarian cancer cells withrelatively similar efficiency, but did not penetrate histologicallymatched normal cell lines to the same degree (FIG. 10A and B). Asingular exception was CCD13-Lu, a cell line derived from lungfibroblasts. The cationic Args was rapidly and efficiently taken up intofibroblasts (FIG. 10A) and all other normal cell lines tested (data notshown).

These observations were confirmed by a more sensitive FACs analyses(FIG. 10C) where p28 fluorescence was about 0.5-6 and p1 8 about 0.5-3fold higher than the corresponding normal cell line, with the exceptionof lung cancer. A similar pattern in intracellular fluorescenceintensity was observed within a histopathologic subtype, melanoma, wherethe relative intensity of p18 was about 50% of that observed with p28(FIG. 10D). Fluorescence intensity over background was also consistentlylower in normal and cancer cell pairs exposed to p18 than p28 (data notshown), again showing less p18 entered individual cells. In all cases,the degree of entry of p18 and p28 into either cancer or normal cellswas significantly less than that observed with Args, where no preferencefor entry was observed (FIG. 10A and B). The predicted Robson structure(data not shown) of p18 suggests that the C-terminal amino acids form apartial f3-sheet. This and the shorter length of p18, which lacks thehydrophilic C-terminal 10 amino acids (amino acids 68-77) of p28, showsthat p18, as a putative PTD for azurin, has a more rapid entry intocancer and normal cells via a non-endocytotic over an endocytotic ormembrane receptor mediated process. MAPAS data {MRS 3.74, MAS 87.1,K_(mpha) 2.37) show that amino acids 69, 70, 75, 76, 85 of azurinprovide the best opportunity for membrane contact, demonstrating thatthe C-terminal region of p28, not present on p18 (amino acids 50-67)contacts specific residues on the cell membrane, irrespective of acell's status.

The preferential penetration of p18 and p28 was confirmed by exposingthe same cell lines to azurin 60-77 (p18b), or amino acids 66-77 (p12),the C-terminal 12 amino acids of p28 (FIG. 11A, B). Here, thepreferential penetration observed with p18 and p28 was completelyabolished. p18b (theoretical pI 4.13) has a short α-helix and partialβ-sheet, and is extremely hydrophilic which together may negatepreferential entry. p12 (theoretical pI 4.33) lacks a secondaryα-helical structure, but is also hydrophilic suggesting overallhydrophilicity may be a major contributor to the decrease in selectivityof cell penetration.

Example 20—Cell Penetration is not a Result of Membrane Disruption

Cell penetration by azurin, p28, and p18 does not result from membranedisruption. An LDH leakage assay using UISO-Mel-2 cells in the presenceof 5-100 μM p28, p18 or azurin (FIG. 12A) suggested that neither peptidenor azurin entered cells by altering plasma membrane integrity (18). Thelack of membrane disruption was confirmed by determining the hemolyticactivity of azurin, p28, and p18 on human erythrocytes against thereceptor mimetic MAP and mast cell degranulating peptide mastoparan 7,which translocates cell membranes as an amphipathic alpha-helix, andactivates heterotrimeric G proteins. Mastoparan 7 caused complete celllysis at 25 μM, while azurin, p28, and p18 had no hemolytic effect whencompared to control (no peptide) (FIG. 12B).

Example 21—p18/p28 Penetration is Energy Dependent and Saturable

The penetration of p28 (FIG. 13A) and p18 (FIG. 13B) into UISO-Mel-2cells is temperature dependent. Cell penetration and intracellulartransport occurs relatively slowly over 3 hr at 4° C., while entry andintracellular transport through various compartments is rapid at 22 and37° C. as p18 and p28 were present in the nucleus of UISO-Mel-2 cellswithin 2 hrs post exposure. The penetration of 5 μM p28 (FIG. 13C) orp18 (FIG. 13D) into UISO-Mel-2 cells after 30 min in the presence of a200 fold excess of unlabeled peptide was severely curtailed, suggestingthat entry was a saturable process and specific receptors or cellsurface proteins or specific residues were, at least in part,responsible for initial entry.

Example 22—Kinetics of p28 and p18

The kinetics of p28 and p18 entry into UISO-Mel-2 cells relative tohuman fibroblasts was calculated after incubation, when cells were fixedand mean fluorescence intensity (MFI) determined. The K_(m) and V_(max)of each peptide were calculated by plotting peptide concentration (μM)vs velocity (MFI/sec) or by Scatchard analysis. Although the penetrationof azurin fragments 50-67 (p18: Vmax 2.46, Km 101.6) and 50-77 tp28:Vmax 1.87, Km 159.1) into cancer and normal cells (Vmax 2.88, Km 102.1and Vmax 1.89, Km 166.0, respectively) differs significantly from eachother, with p18 entering −42% faster, the rate of the entry of eachpeptide into normal and cancer cells is virtually identical. Theincrease in amount of fluorescence following exposure of cancer cells top28 relative to p18 is likely due to the increase in the amount of p28entering malignant cells. ¹²⁵I azurin and p18 bound to UISO-Mel-2 cellswith a similar affinity. In contrast, significantly more p28 (K_(d) 2.5μm, Bmax 3.0 pm) bound to UISO-Mel-2 cells with a higher affinity whenexposed for a longer period of time (20 min vs 2 min) at a highertemperature (37° C. vs 4° C.) than either p18 (K_(d) 18 min, Bmax 0.51pm) or azurin (K_(d) 10 nm and 0.48 pm). These results show that azurin,p28, and p18 all bind with relatively high affinity and capacity to asite on the cancer and normal cell surface prior to entry, but may entervia more than one mechanism.

Example 23—p18/p28 Penetration Involves Caveolae and the Golgi Complex

As a class, cationic CPPs such as pTat and Arg₈ (SEQ ID NO: 95) entercells by initially binding to anionic, sulfated proteoglycans prior toendocytosis. Incubation of p28 and p18 and Arg₈ (SEQ ID NO: 95) withUISO-Mel-2 cells under serum free conditions in the presence/absence of100 μg/ml heparin sulfite (HS) significantly reduced the amount ofintracellular Arg₈ (SEQ ID NO: 95), but did not alter the entry ofeither p28 or p18 (FIG. 14A). The penetration of p18 and p28 intoUISO-Mel-2 cells in the presence or absence of a specific inhibitor ofO-linked glycosylation, BnGalNac, and neruaminidase, which cleavessialic acid residues, was further characterized (FIG. 14B), and noinhibition of penetration was observed. However, tunicamycin, aninhibitor of N-linked glycosylation, significantly reduced thepenetration of p18 and p28 across the cell membrane.

The entry of p18 and p28 into UISO-Mel-2 cells was also analyzed usinginhibitors of energy dependent transport mechanisms, i.e., ATP. Sodiumazide (FIG. 14B) and ouabain (Na⁺K⁺ ATPase pump) did not significantlyinhibit the penetration of either peptide suggesting non endocytosicpathways might also be involved in the penetration of these peptides.Chlorpromazine (CPZ), a specific inhibitor of clathrin mediatedendocytosis, also had no effect on penetration, nor did themacropinocytosis inhibitor amiloride. (FIG. 14B). Stabilization ofmicrotubules with taxol had no effect on penetration, but disruption ofactin filaments and macropinocytosis with Cytochalasin D produced asmall (−20%), reproducible inhibition of the penetration of p18 and p28.The lack of effect of amiloride suggests that the inhibitory activity ofCytochalasin D is probably through its effect on actin filaments.

Inhibition of the cell cycle with staurosporine did not blockpenetration, suggesting that penetration was not cell cycle specific.The lack of effect of staurosporine on p18 and p28 penetration of thecancer cell plasma membrane also suggests that a Src kinase/tyrosinekinase dependent pathway was not involved in penetration, was dynaminindependent, and hence independent of caveolae budding. Neither p18 norp28 co-localized with flotillin-1 (data not shown) a protein thatresides within the plasma membrane and in a specific population ofendocytic intermediates, again arguing against a role for flotillin anddynamin in internalization. In contrast, nocodazole, which disruptscaveolae transport and inhibitors of cholesterol mobilization and hence,caveolae-mediated endocytosis, inhibited penetration 50-65%.

The intracellular disposition of p18 and p28 was then analyzed usingwortmannin, an inhibitor of early endosome formation, monensin, whichinhibits late endosome/lysosome, and brefeldin A (BFA), a disrupter ofthe Golgi apparatus. Wortmannin did not block the intracellularaccumulation of either p18 or p28 suggesting that, unlike cholera toxin,a caveolae to early endosome pathway is not involved in theintracellular trafficking of p18 and p28. The lack of early endosomeinvolvement in the intracellular trafficking of p18 and p28 alsosuggests that clathrin mediated endocytosis is not involved ininternalization of these peptides.

However, monensin (FIG. 14B) and BFA reduced the intracellularaccumulation of both peptides with a greater inhibitory effect on p28(˜30%) than p18 (˜10%) (FIG. 14B). The penetration of p28 and p18 intofibroblasts was also inhibited by MβCD, nocodazole, monensin andtunicamycin, but not by amiloride, sodium azide, and CPZ (FIG. 14C).This shows that at least one mechanism of entry into cancer and normalcells is similar, but additional preferential accumulation into cancercells may be a function of the number of common membrane receptors orstructures, i.e., caveolae (FIG. 14D, panels 1, 2). Alexafluor 568labeled p18 and p28 co-localized with caveolin-1 and golgin 97antibodies (FIG. 14D panels 1,2). This confirms that these organellesare involved in the intracellular trafficking of p18 and p28.Interestingly, azurin, but neither p18 nor p28 colocalized withmitochondrial specific fluorescence (FIG. 14D panel 3). In contrast, p28and azurin, but not p18, co-localized with lysosomes (FIG. 14D panel 4).

Example 24—Functional Analysis of p28 and p18

Azurin inhibits the growth of several human cancer cell lines in vitroand in vivo. FIGS. 15A and B illustrate the effect of p18 and p28relative to azurin and dacarbazine (DTIC) on UISO-Mel-2 cells asdetermined by MTT and cell count. After 72 hrs exposure, azurindecreased (p<0.5) cell survival at 100 and 200 μM-15% (FIG. 15A). p28had inhibited cell survival 14 and 22% (p<0.05) at 100 and 200 μM,respectively. In contrast, p18 had no effect, while dacarbazine (DTIC)produced a significant dose-related decrease on UISO-Mel-2 survival.Azurin and p28 (200 μM) also significantly decreased the survival ofUISO-Mel-23 and 29 cells. p18 had no effect on UISO-Mel-2 cellproliferation. The apparent increase (˜30-35%; UISO-Mel-2) in p28 andazurin inhibition of melanoma cell proliferation, as measured by directcell counting, suggests that the inhibitory effect may reside primarilyat the level of cell cycle with apoptosis subsequent to any delay.Although p18 penetrated cancer cells preferentially, unlike p28, it hadvirtually no inhibitory activity on cell proliferation. This resultdemonstrates that the cytostatic and cytotoxic activity of p28 lies inthe C-terminal 10-12 amino acids of the sequence.

Example 25—Azurin and p28 Binding and Entry into Cells

UISO-Mel-2 or fibroblast cells (3×10⁵ cells) were suspended in MEMEmedia without phenol red. Reactions were started by adding Alexafluor568-conjugated p28 at 10, 50, 100, 150, 250, 300 and 400 μM for 30, 60,90 and 120 sec on ice. Cells were analyzed by flow cytometry. The uptakeof the peptides into the cells are shown in the graphs of FIG. 17A. TheKm and Vmax were calculated by plotting peptide concentration (μM) vsvelocity (MFI/sec). These calculations are depicted in FIG. 17B. Peptidebinding and entry was determined using whole Mel2 cells (50,000cells/ml), were incubated for 30 min at 37° C. with increasingconcentrations (0-175 nM) of radiolabeled azurin in the presence/absenceof 1000 fold excess of unlabeled p28, or azurin, and radioactivityremaining in the cell pellet counted using a gamma counter. The resultsare depicted in FIG. 17C. Radioactivity in cells incubated with ¹²⁵Iazurin alone was considered total binding; radioactivity in the presenceof unlabeled azurin or p28 was considered nonspecific binding. Specificbinding was determined by subtracting nonspecific binding from totalbinding and Scatchard plots generated.

Example 26—Inhibition of Cancer Growth Through p53 Using Azurin-DerivedPeptides: Materials and Methods

Cell culture.

Human breast cancer cell lines, MCF-7 (p53 wt), obtained from ATCC(Manassas, Va.) and MDD2 (p53 dominant negative) from Dr. Andrei V.Gudkov (Lerner Research Institute, Cleveland, Ohio) were cultured inMEM-E (Invitrogen, Carlsbad, Calif.) containing 2 mM L-glutamine, 0.1 mMessential amino acids supplemented with 10% heat inactivated fetalbovine serum, 100 Units/ml penicillin and 100 μg/ml streptomycin.

Bacterial Culture and Isolation of Azurin.

Escherichia coli JM109 was used as the host strain for production ofwild type azurin. Culture conditions and protein purification steps wereas described in Yamada, et al., Infect Immun, 70:7054-7062 (2002) andGoto, et al., Mol Microbiol, 47:549-449 (2003).

Peptide Synthesis.

All azurin-derived peptides including p18, Leu⁵⁰-Gly⁶⁷LSTAADMQGVVTDGMASG (SEQ ID NO: 25), p28 Leu⁵⁰-Asp⁷⁷ (SEQ ID NO: 2)LSTAADMQGVVTDGMASGLDKDYLKPDD, p18b Val⁶⁰-Asp⁷⁷ (SEQ ID NO: 34)VTDGMASGLDKDYLKPDD, p12 Gly⁶⁶-Asp⁷⁷ SGLDKDYLKPDD (SEQ ID NO: 35), andpoly arginine (Arg₈) (SEQ ID NO: 95) were synthesized by CS Bio, Inc.(Menlo Park, Calif.) as >95% purity and mass balance.

Proliferation Assays.

Cells were seeded in MEM-E in quadruplicate into 24-well plates (BectonDickinson, Franklin Lakes, N.J.) at a density of 12×10³ cells/well andincubated in the presence of 5, 50, 100 and 200 μM p28 for 24, 48 and 72hr. Media was changed daily. Control wells received MEM-E without p28 (8replicates). Doxorubicin (10 μM) was used as positive control (Z1coulter; Beckman Coulter Inc., Fullerton, Calif.). Values represent (%)of control. Significance between control and treated groups wasdetermined by Student's t-test.

MTT Assay:

MCF-7 cells were seeded at a density of 2000 cells/well (quadruplicate)allowed to attach for 24 hrs, and freshly prepared peptide (10 μl) orMEM-E added to each well. After 24 hrs, medium and p18, p28, azurin ordoxorubicin were added daily. After incubation, 10 μl of MTT reagent(Trevigen, Gaithersburg, Md.) was added to each well, the samplesincubated for 3 hr at RT, 100 μl of detergent added to each well, andincubated for an additional 3 hr at 37° C. Absorbance (570 mm) wasmeasured (SpectraMax 340 plate reader, Molecular Devices Corporation,Sunnyvale, Calif.) and percent change in treated cells determined.Significance p<0.05) between control and treated groups was determinedby Student's t-test.

Xenograft Model.

Estradiol pre-treated (0.72 mg/pellet, 60-day release; InnovativeResearch, Sarasota, Fla.) female athymic mice (Harlan; 4-5 weeks old)received 3×10⁶ MCF-7 cells s.c. in the right flank and randomized intocontrol and experimental groups prior to treatment. Control animalsreceived PBS/castor oil i.p. Paclitaxel, 15 μmmol/kg in PBS/castor oilwas injected i.p. on days 10, 14, 21 and 25 post-tumor cell inoculation,or p28, 5 or 10 mg/kg in sterile PBS i.p. daily was injected for 30days. Tumor volume was determined 3×/week. Body weights were measuredtwice weekly. Mice were necropsied on day 31 and all tumors collectedfor histopathology and immunocytochemistry. Significance (p<0.05)between control and treated groups was determined by Student's t-test.

Immunocytochemistry.

BrdU, 50 mg/kg body wt, was injected i.p., 2 hrs prior to necropsy.Tumor cell nuclei labeled with BrdU were identified with an anti-BrdUmonoclonal antibody (Beckon Dickinson, Franklin Lakes, N.J.). p53expression was quantified in formalin fixed, 5μ paraffin sectionstreated with 10 mM citrate buffer in a pressure cooker for 6 min. Cooledslides were treated with 3% H₂O₂ for 10 min to block endogenousperoxidase, covered with blocking serum for 10 min, and exposed to p53antibody (DO-1, Santa Cruz Biotechnology, Santa Cruz, Calif.) for 2 hrsat room temperature. Rat anti-mouse IgG2a was used as the secondantibody. Cells expressing p53 were identified using a Vectastain EliteABC kit (Vector Laboratories, Burlingame, Calif.) and3,3′-diaminobenzidine tetrahydrochloride (Sigma Aldrich, St. Louis,Mo.). Slides were counterstained with hematoxylin. Ten non-overlappingfields (250 cells/field) from each tumor periphery were screened (40×)for p53 labeled cells.

Confocal Microscopy:

Cells were seeded overnight on glass cover slips at 37° C. under 5% CO₂,rinsed with fresh media, and incubated at 37° C. for 2 hr in pre-warmedmedia containing Alexa Fluor 568 labeled peptides (20 μM), Arg₈ (SEQ IDNO: 95) (5 μM), or media alone. After incubation, cover slips wererinsed 3× with PBS, fixed in 2.5% formalin for 5 min, washed 2× in PBS,once in d.i.H₂O, and mounted in media containing 1.5 μg/ml DAPI tocounter stain nuclei (VECTASHIELD®, Vector Laboratories). Cyclin B1 andp21 staining were determined in fixed cells, permiabilized by methanoland acetone, washed with PBS and incubated with anti-p21 or cyclin B ata 1:200 dilution (Santa Cruz Biotechnology). Secondary antibodyconjugated Alexa Fluor 568 was used at 1:100 dilution. Cellular uptakeand intracellular distribution were determined using an invertedconfocal laser scanning microscope (Model LC510, Carl Zeiss Inc.,Gottingen, Germany).

Kinetics:

MCF-7 and MDD2 cells (3×10⁵ cells) were suspended in MEM-E withoutphenol red. Reactions were started by adding Alexa Fluor 568-conjugatedp28 at 1, 10, 25, 50, 100, 150 and 200 μM for 30, 60, 90 and 120 sec onice. After incubation, 1 ml of cold-PBS was added to the reactionmixture and cells centrifuged 2× at 600×g for 2 min at 4° C. At least10,000 fixed cells were analyzed for each time point and concentrationby flow cytometry and their background and relative fluorescencecalculated.

Cell Cycle Analysis.

MCF-7 and MDD2 cells were incubated with 50 μM of p28 for 48 and 72 hrat 37° C., washed twice with phosphate-buffered saline (PBS) and fixedwith 70% ethanol at −20° C. Fixed cells were washed twice with PBS andstained by 50 μg/ml of propidium iodide (PI) in PBS containing 20 μg/mlof RNase A. Flow cytometry (EPICS Elite ESP, Beckman Coulter, Fullerton,Calif.) was used to determine DNA content. A minimum of ten thousandcells were collected in each experiment.

Immunoblotting.

MCF-7 and MDD2 cells were cultured with 50 μM p28 for 0, 24, 48 and 72hr. and whole cell lysates prepared according to the methods describedearlier (3). Cell lysates for phosphorylated cdc2 (p-cdc2) was preparedin 10 mM NaF, 137 mM NaCl, 1 mM NaVO₄, 10 mM EDTA, 1% NP-40, 1 mM DTTand proteinase inhibitors (Sigma Aldrich). Antibodies against p53, p27,CDKs, cyclins (Santa Cruz Biotechnology), p21 (Invitrogen) were usedaccording to the suppliers' instructions. Actin expression wasdetermined with a monoclonal actin antibody (Santa Cruz Biotechnology)and protein bands visualized using ECL reagent (Santa CruzBiotechnology).

Anti-p28 Antibody.

A cysteine was introduced at the N-terminus of p28 (CS Bio Inc., MenloPark, Calif.), and then the peptide was conjugated with Keyhole limpethemocyanin through the thiol groups of the cysteine residue, the complexwas inoculated intradermally and subcutaneously, and a polyclonalantibody specific for 11-28 amino acids of p28 (amino acids 60-77 ofazurin) in rabbits (New Zealand White, Covance, Mich.) was generated.Antibody titer was determined by direct ELISA using p28 (0-3 μg/well).An antibody dilution of 1:140,000 was sufficient to give a reproduciblechange in absorbance of 0.5 at 450 nm after 15 min incubation withsubstrate (1-Step PNPP, Pierce, Rockford, Ill.) at 25° C., when 96well-plates (Nunc, Rochester, N.Y.) were coated with 1 μg/well p28.

GST Pull-Down Assay.

p28 binding to p53 was assayed using a GST pull down assay essentiallyas described in Punj, et al, Oncogene 23:2367-2378 (2004). PurifiedGST-p28 (10 and 20 μg/reaction), GST-MDM2 (20 μg/reaction) and GST alone(20 μg/reaction) were bound to Glutathione Sepharose 4B beads (GEHealthcare, N.J.) and unbound peptide removed by washing 2× with PBS.Whole cell lysates of MCF-7 cells were generated with PBS/0.1% TritonX-100 containing proteinase inhibitor cocktail (Sigma-Aldrich) on icefor 15 min, and centrifuged at 14000 r.p.m. for 30 min at 4° C.Resultant supernatants were mixed with beads, incubated for 2 hr at 4°C., washed 2× with PBS to remove unbound cell lysate and then boiled inSD S-sample buffer prior to loading on 10% SDS-PAGE. Membranes wereincubated with skim milk (5%) in TBST (Tris/0.05% Tween20) andpolyclonal p53 antibody (FL-393, Santa Cruz Biotechnology) in 5% skimmilk at 4° C., washed 3× with TBST, secondary rabbit IgG-HRP antibody(Sigma-Aldrich) added, incubated for 1 hr at room temperature (r/t), andwashed 3× with TBST.

Potential binding sites on p53 were identified as follows. Interactionat the MDM2 binding site (amino acids 18-23) of p53 was analyzed using aGST-pull down assay in the presence of p28 (10-50 molar excess) and p53bands detected by immunoblotting (IB). Three different anti-p53antibodies, Pab 1801 (32-79 amino acids; Santa Cruz Biotechnology), ab2433 (277-296-amino acids; Abeam Inc., Cambridge, Mass.) and Pab1802(306-393-amino acids; Santa Cruz Biotechnology), that represent thebroadest coverage of the p53 protein available, were each reacted withGST-p53 immobilized beads in the presence of p28. After incubation,samples were washed 2× with PBS to remove unbound p28, boiled in nativePAGE sample buffer (Tri/glycerol/BPB) and loaded on 5% Native-PAGE.Samples were transferred to PVDF membrane by electroblotting (0.2 Ampfor 1 hr), membranes blocked with skim milk (5%) in TBST and incubatedwith a polyclonal antibody to p28 (1:5000 dilution) in 5% skim milk at4° C. After washing with TBST, HRP-conjugated rabbit anti-IgG antibody(1:7000 dilution, Santa Cruz Biotechnology) was applied. p28 bands werevisualized using ECL reagent. Binding domains on p28 were identifiedusing a competition assay between p28 and the p28 fragments p12, p18 andp18b for GST-p53 (20 μg/reaction) immobilized on Glutathione Sepharose4B beads. Reactions were incubated for 2 hr at 4° C., washed 2× with PBSto remove unbound p28, then boiled in native PAGE sample buffer(Tri/glycerol/BPB) and loaded on 5% Native-PAGE. Proteins weretransferred to a PVDF membrane by electro blotting (0.2 Amp for 1 hr),blocked, and incubated with the polyclonal antibody to p28 at 4° C. for16 hr. p28 bands were visualized with ECL reagent. Band intensity wasdetermined using Gel & Graph Digitizing Software, UN-SCAN-IT™ (SilkScientific Inc., Orem, Utah) and the ratio of specific protein/actincalculated. Numbers displayed below each protein band are relativepercentage of the protein babd intensity immediately prior to treatment(0 hr expressed as 100%).

p53 DNA-Binding Activity.

Nuclear fractions (Nuclear Extraction kit, Active Motif, Carlsbad,Calif.) were isolated from MCF-7 cells after incubation with either 50μM p28 or azurin at for 24 h according to the manufacturers'instructions. Nuclear extract supernatants were collected bycentrifugation at 14,000 rpm for 10 min at 4° C. Protein concentrationswere determined using the Bradford method. DNA-binding activity of p53was measured using a TransAM p53 kit (Active Motif). Briefly, 40 μl ofbinding buffer containing DTT and poly[d(I-C)] was introduced to eachwell to prevent non-specific binding to the p53 consensusoligonucleotide. Nuclear extracts were applied to each well, withH₂O₂-treated or buffer only as positive and negative controls,respectively, and incubated 1 hr r/t. Wells were washed 3× and 100 μl ofp53 antibody (1:1000 dilution) applied and incubated at r/t for 1 hr.After washing, secondary antibody conjugated with HRP was added, samplesincubated for 1 hr and developed for 3 min in the dark. p53 binding toDNA was determined by absorbance at 450 and 655 nm.

Example 27—Effect of p28 Treatment on the Growth of Human Cancer CellsIn Vitro and In Vivo

Azurin exerts its anti-cancer activity through induction of ap53-mediated apoptosis. FIGS. 54 A and B show the effect of p28 anddoxorubicin on wt p53 (positive) MCF-7 cells as determined by directcell count and MTT assay. p28 initially inhibited the proliferation ofMCF-7 cells in vitro (FIG. 54A) in a dose and time related mannerproducing a significant decrease (p<0.05) in cell number ˜23% at 5 μMand 36% at 50-200 μM after 24 hr exposure. Doxorubicin (DNAintercalating agent) also significantly inhibited cell growth intime-dependent manner. Cell survival determined by MTT assay was notsignificantly altered by p28, while doxorubicin exhibited a significanttime related decrease in MCF-7 cell survival (FIG. 54B). p28 alsoproduced a significant dose related decrease in the volume ofxenografted MCF-7 cells in athymic mice over a daily, 30-day i.p.exposure (FIG. 54C), decreasing tumor volume (p<0.05) to that observedwith Paclitaxel®, without inducing either a loss in body weight orbehavioral change. By day 30, 10 mg/kg p28 daily i.p. inhibited MCF-7growth to a greater extent (˜20%) than 15 μmol/kg Paclitaxel® on days10, 14, 21 and 25 post-tumor cell inoculation. The reduction in BrdUlabeling associated with the p28-induced decrease in tumor volumesuggested cell cycle was inhibited (Table 5). In contrast, the reductionin BrdU labeling and tumor volume was accompanied by a slight increasein nuclear p53-staining in p28 and a significant increase in thePaclitaxel® treated group compared to control (Table X).

TABLE 5 BrdU and p53 in MCF-7 xenograft tumors N BrdU (%) p53 (%)Control 7 21.0 ± 2.7  15.6 ± 0.82 p28 (5 mg/kg) 4 17.6 ± 0.75* 15.8 ±0.51 p28 (10 mg/kg) 3 16.1 ± 1.4*  17.7 ± 0.92 Paclitaxel 6  9.0 ± 1.8** 25.4 ± 0.65** All tumors were collected on day 31 post treatment.Values represent Mean ± SEM. *p < 0.025; **p < 0.01 from respectivecontrol; student's T-test.

Example 28—Inhibition of Cell Cycle Progression by p28

Cell cycle analysis of the two isogenic breast cancer cell lines, MCF-7(p53 wt) and MDD2 (p53 dominant negative), revealed an increased cellpopulation at the G₂/M phase after exposure to p28 for 48-72 hrs andsubsequent induction of apoptosis at 72 hrs in MCF-7 cells (FIG. 55A).There was essentially no inhibition of cell cycle progression orapoptosis in p28-treated MDD2 cells (FIG. 55B). The lack of cell cycleinhibition and apoptosis in p28-treated MDD2 cells (FIG. 55B) was notdue to a difference in p28 entry into MDD2 cells (FIG. 55C) ordifference in V_(max) (MCF-7: 1.83 MFI/sec, MDD2: 2.21 MFI/sec) or K_(m)(MCF-7: 144.3 μM, MDD2: 147.9 μM).

Example 29—p53 Levels are Elevated by p28

Azurin forms a complex with p53 and elevates intracellular p53 levels inMCF-7 cells. The intracellular level of p53 in MCF-7 cells alsosignificantly increased with time post exposure to p28 (FIG. 56). A GSTpull-down assay suggested p28 binds to p53 (FIG. 56B). Here, GST-p28 andGST-MDM2 successfully pulled down p53 from MCF-7 cell lysates, but GSTalone did not. Molar increases of p28 did not compete for binding withGST-MDM2 (FIG. 56C) suggesting that amino acids 18-23 of p53 were not apreferred binding site for p28. An additional GST-pull down assay in thepresence or absence of p53 antibodies, which recognize different motifsof the p53 protein (amino acids 32-79, 277-296 and 306-393), did notblock p28 binding to p53, suggesting that p28 binds to a region of p53outside these recognition sites (FIG. 56C).

When Sepharose 4B-glutathione beads immobilized with GST-p53 proteinwere incubated with p28 and either amino acids 66-77, amino acids 50-67,or amino acids 60-77 of azurin, (p28 fragments p12, p18 and p18b)respectively, significant amounts of p28 were displaced by p18 and p18b,but only weakly when p12 was used as the competitor (FIG. 56D). Theseresults suggest that maximal binding to p53 occurs within amino acids11-28 of p28 (amino acids 60-77 of azurin).

As p28 enhances intracellular levels of p53, the DNA-binding activity ofp53 obtained from MCF-7 cell nuclear extracts treated with p28 or azurinwas also examined. p53 DNA-binding activity in the nuclear fraction ofMCF-7 cells treated by p28 and azurin was 1.8 and 2.3 fold higher thancontrol (p>0.1, p28 vs azurin). The p53 wt consensus, but not themutated oligonucleotide sequence, completely blocked the p28 inducedincrease in p53, confirming that the p53 in nuclear extracts of MCF-7cells binds specifically to the consensus oligonucleotide sequence forwt p53 (FIG. 56E).

Example 30—Modulation of Cell Cycle Related Proteins by p28

Upregulation of the CDK inhibitors (CDKIs), p²¹ and p27, blocks cellcycle progression. p28 increased intracellular levels of p21, p27, CDK6and cyclin B1 over control in MCF-7 cells with time post-exposure (FIG.57A). The levels of CDK2 and cyclin A, essential proteins in the mitoticprocess, subsequently decreased with time post-exposure in p28 treatedMCF-7 cells (FIG. 57A). In contrast, p53, cdc2, CDK2, CDK4 and CDK6essentially remained constant in MDD2 cells (FIG. 57B), while cyclin Aand cyclin B1 (48 hrs) increased slightly. Since p21 can be expressed bya p53-independent pathway in MDD2 cells, p21 remained detectable. p28did not alter the level of p21, however (FIG. 57B). In contrast, p27 wasnot detectable in untreated or p28 exposed MDD2 cells. The increasedlevels of p21 and cyclin B1 in MCF-7 cells detected by immunoblotting inresponse to p28 are reflected by their increase in nuclear and cytosoliccompartments, respectively (FIGS. 57C and D). Exposure of MCF-7 cells top28 also induced the accumulation of phosphorylated cdc2 (p-cdc2), theinactive form of cdc2. The level of p-cdc2 did not increase followingexposure of MDD2 cells to p28 (FIG. 57E).

Example 31—Imaging p18 and p28 Entry into Mouse Organs

Small animal in vivo imaging has important significance in biologicalstudies, including human cancer research. The ability to track andvisualize a tagged biological probe allows researchers to visualizebiological processes and deduce mechanisms of action and efficacy.Imaging can be used to directly visualize trafficking of near infraredlabeled peptides of the cupredoxin class of proteins, including azurinand the azurin fragments p28 and p18, to primary and metastatic tumorsites in xenograft bearing nude mice. J Biomed Optics 10:054010-1-11,2005; J Amer Soc Exp Neuother 2:215-225, 2005; Topics Curr Chem222:1-29, 2002

Procedure.

Athymic nude mice bearing Mel2 xenograft tumors were monitored untiltumor size reached 0.5 cm³. Mice were anesthetized using a mixture of2:1 ketamine:xylazine; recommended dosage is 10 μl/gm mouse b.w. s.c.Anesthetized mice were scanned directly before and after injection oflabeled peptide with an iCor Odyssey Imager. Anesthetized mice wereinjected i.v. (tail vein) with 100 μl of IRDye™ 800 cw labeled p18/p28at a concentration of 1.25 μg/μl-125 μg per mouse. Mice were scanned atleast once every 24 hours until excess dye cleared their system(generally ˜5 days). On the fifth day, mice were sacrificed andindividual animals scanned a final time. Organs, including the kidneys,stomach, intestine, spleen, brain, heart, and lungs, and tumors wereexcised, split in half, and half were fixed for histologicalexamination. The other half of the organs and tumors was covered with asmall amount of PBS, and then scanned.

What is claimed is:
 1. A method for treating a patient at risk for ordiagnosed with cancer comprising: administering to a mammalian patient apharmaceutical composition comprising one or more of a peptide selectedfrom the group consisting of: SEQ ID NO: 36 and SEQ ID NO: 37 and apharmaceutically acceptable carrier; and wherein the pharmaceuticalcomposition is administered by a mode selected from the group consistingof intravenous injection, intramuscular injection, subcutaneousinjection, inhalation, topical administration, transdermal patch,suppository, vitreous injection, and oral administration.
 2. The methodof claim 1, wherein the patient is human.
 3. The method of claim 2,wherein the patient is at a higher risk to develop cancer than thegeneral population.
 4. The method of claim 3, wherein the cancer isselected from the group consisting of melanoma, breast, pancreas,glioblastoma, astrocytoma, lung, colorectal, neck and head, bladder,prostate, skin, and cervical cancer.
 5. The method of claim 2, whereinthe patient has at least one high risk feature.
 6. The method of claim2, wherein the patient has premalignant lesions.
 7. The method of claim1, wherein the mode of administration is by intravenous injection. 8.The method of claim 1, wherein the pharmaceutical composition furthercomprises a cargo compound.